Agonist antibodies

ABSTRACT

Various forms of c-mpl agonist antibodies are shown to influence the replication, differentiation or maturation of blood cells, especially megakaryocytes and megakaryocyte progenitor cells. Accordingly, these compounds may be used for treatment of thrombocytopenia.

This application is a non-provisional application filed under 37 CFR1.53(b), claiming priority under 35 USC 119(e) to provisionalapplication No. 60/056,736, filed Aug. 22, 1997, the contents of whichare incorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to the recombinant synthesis and purification ofprotein antibodies that influence survival, proliferation,differentiation or maturation of hematopoietic cells, especiallyplatelet progenitor cells and to antibodies that influence the growthand differentiation of cells expressing a protein kinase receptor. Thisinvention also relates to the cloning and expression of nucleic acidsencoding antibody ligands (thrombopoietin receptor agonist antibodies)capable of binding to and activating a thrombopoietin receptor such asc-mpl, a member of the cytokine receptor superfamily. This inventionfurther relates to the use of these antibodies alone or in combinationwith other cytokines to treat immune or hematopoietic disordersincluding thrombocytopenia and to uses in assays.

BACKGROUND OF THE INVENTION

In 1994 several groups reported the isolation and cloning ofthrombopoietin (F. de Sauvage et al., Nature 369:533 (1994); S. Lok etal., Nature 369:565 (1994); T D. Bartley et al., Cell 77:1117 (1994); Y.Sohma et al., FEBS Letters 353:57 (1994); D J. Kuter et al., Proc. Natl.Acad. Sci. 91:11104 (1994)). This was the culmination of more than 30years of research initiated in the late 50's when Yamamoto (S. Yamamoto,Acta Haematol Jpn. 20:163-178. (1957)) and Kelemen (E. Kelemen et al.,Acta Haematol (Basel). 20:350-355 (1958)) proposed that physiologicalplatelet production is controlled by a humoral factor termed“thrombopoietin”(TPO). Although routinely detected in urine, plasma andserum from thrombocytopenic animals and patients, as well as kidney cellconditioned media, purification of TPO proved to be a daunting task (fora review see M S. Gordon et al., Blood 80:302 (1992); W. Vainchenker etal., Critical Rev. Oncology/Hematology 20:165 (1995)). In the absence ofpurified TPO and the apparent fact that numerous plieotrophic cytokinesaffected megakaryocytopoiesis (M S. Gordon et al., Blood 80:302 (1992);W. Vainchenker et al., Critical Rev. Oncology/Hematology 20:165 (1995)),the existence of a lineage specific factor that regulated plateletproduction was doubted until the discovery of the orphan cytokinereceptor c-Mpl in 1990 (M. Souyri et al., Cell 63:1137 (1990); I. Vigonet al., Proc. Natl. Acad. Sci. 89:5640 (1992)). The expression of c-Mplwas found to be restricted to progenitor cells, megakaryocytes andplatelets, and c-Mpl antisense oligonucleotides selectively inhibited invitro megakaryocytopoiesis (M. Methia et al., Blood 82:1395 (1993)).From this it was postulated that c-Mpl played a critical role inregulating megakaryocytopoiesis and that its putative ligand may be thelong sought TPO (M. Methia et al., supra). Following this discoveryseveral groups utilizing c-Mpl ligand specific cell proliferation assaysand c-Mpl as a purification tool isolated and cloned the ligand forc-Mpl (F. de Sauvage a al., supra; S. Lok et al., supra; T D. Bartley etal., supra). In addition two other groups independently reported thepurification of the Mpl-ligand using standard chromatography techniquesand megakaryocyte assays (Y. Sohma et al., supra, D J. Kuteret al.,supra). In the years since its reported discovery numerous studiesclearly indicate that the Mpl-ligand possess all the characteristicsthat have long been attributed to the purported regulator ofmegakaryocytopoiesis and thrombopoiesis and consequently, is nowreferred to as TPO. The Mpl ligand is currently referred to as eitherTPO or as megakaryocyte growth and differentiation factor (MGDF).

Human TPO consists of 332 amino acids that can be divided into 2domains; an amino terminal domain of 153 amino acids showing 23%identity (50% similarity) to erythropoietin (EPO) and a unique 181 aminoacid C-terminal domain that is highly glycosylated ((F. de Sauvage etal., supra; S. Lok et al., supra; T D. Bartley et al., supra). TheEPO-like domain of TPO contains 4 cysteines, 3 of which are conservedwith EPO. The first and last and the two middle cysteines form twodisulfide bridges, respectively, which are both required for activity(T. Kato et al., Blood 86 (suppl 1):365 (1995)). None of the Asn-linkedglycosylation sites present in EPO are conserved in the EPO-like domainof TPO, however, the EPO-like domain of recombinant TPO (rTPO) contains2-3 O-linked glycosylations (M. Eng et al., Protein Science 5(suppl1):105 (1996)). A recombinant truncated form of TPO (rTPO153),consisting of only the EPO-like domain, is fully functional in vitro,indicating that this domain contains all the required structuralelements to bind and activate Mpl (F. de Sauvage et al., supra; D L.Eaton et al., Blood 84(suppl 1):241 (1994)). The carboxy terminal domainof TPO contains 6 N-linked and 18 O-linked glycosylated sites and isrich in proline, serine and threonine (M. Eng et al., supra). Thefunction of this domain remains to be elucidated. However, because ofits high degree of glycosylation this region may act to stabilize andincrease the half life of circulating TPO. This is supported by theobservation that rTPO153 has a half life of 1.5 hours compared to 18-24hours for full length glycosylated rTPO (G R. Thomas et al., Stem Cells14(suppl 1) (1996).

The two domains of TPO are separated by a potential dibasic proteolyticcleavage site that is conserved among the various species examined.Processing at this site could be responsible for releasing theC-terminal region from the EPO domain in vivo. The physiologicalrelevance of this potential cleavage site is unclear at this time.Whether TPO circulates as an intact full length molecule or as atruncated form is also equivocal. When aplastic porcine plasma wassubjected to gel filtration chromatography, TPO activity present in thisplasma resolved with a Mr. of ˜150,000 ((F. de Sauvage et al., supra).Purified full length rTPO also resolves at this Mr., whereas thetruncated forms resolve with Mr. ranging from 18,000-30,000. Using TPOELISAs that selectively detect either full length or truncated TPO ithas also been shown that full length TPO is the predominant form in theplasma of marrow transplant patients (Y G. Meng et al., Blood 86(suppl.1):313 (1995)).

Prior to the discovery of c-Mpl and the isolation of TPO, it was thoughtthat megakaryocytopoiesis was regulated at multiple cellular levels (MS. Gordon et al., supra; W. Vainchenker et al., supra; Y G. Meng et al.,supra). This hypothesis was based on the observation that certainhematopoietic growth factors stimulated proliferation of megakaryocyteprogenitors while others primarily affected maturation (M S. Gordon etal., supra; W. Vainchenker et al., supra; Y G. Meng et al., supra).Other data indicated that plasma from thrombocytopenic animals containeddistinct activities that either affected proliferation (meg-CSF) ormaturation (TPO) of megakaryocytes (R J. Hill et al., Exp. Hematol20:354 (1992)). Wendling and her colleagues (F. Wendling et al., Nature369:571 (1994)) initially dispelled this theory by demonstrating thatall the megakaryocyte colony-stimulating and thrombopoietic activitiesin thrombocytopenic plasma could be neutralized by soluble Mpl. Thisindicated that these activities are due to a single factor, theMpl-ligand. Numerous studies have now shown that recombinant forms ofTPO not only induce proliferation of progenitor megakaryocytes but alsotheir maturation (K. Kaushansky et al., Nature 369:568 (1994); F C.Zeigler et al., Blood 94:4045 (1994); V C. Broudy et al., Blood 85:1719(1995); J L. Nichol et al., J. Clin. Invest. 95:2973 (1995); N. Banu etal., Blood 86:1331 (1995); N. Debili et al., Blood 86:2516 (1995); P.Angchaisuksiri et al., Br. J. Haematol. 93:13 (1996); E S. Choi et al.,Blood 85:402 (1995)). Human CD34+, CD34+,CD41+ cells (F C. Zeigler etal., supra; V C. Broudy et al., supra; J L. Nichol et al., supra; N.Banu et al., supra;) or purified murine stem cells (sca+,lin−, kit+) (K.Kaushansky et al., supra: F C. Zeigler et al., supra) cultured with rTPOselectively differentiate to megakaryocytes. rTPO induces thedifferentiation and proliferation of megakaryocyte colonies in semisolidcultures and single megakaryocytes in liquid suspension cultures. Thisactivity appears to be a direct effect of TPO as limiting dilutionexperiments show a direct correlation between progenitors seeded andmegakaryocytes obtained (N. Debili et al., supra). In additioncomparable results are obtained in serum free or serum containingculture conditions (N. Banu et al., supra; N. Debili et al., supra; P.Angchaisuksiri et al., supra;). These observations indicate that neitheraccessory cells or serum components are required for TPO to inducemegakaryocyte growth and differentiation in vitro.

The effect of rTPO on the megakaryocyte maturation process is dramatic.rTPO induces highly purified murine or human progenitor cells in liquidculture to differentiate into very large mature polyploid megakaryocytes(F C. Zeigler et al., supra; V C. Broudy et al., supra; J L. Nichol etal., supra; N. Debili et al., supra). Megakaryocytes from such culturesexhibit ploidy of 4N-16N with ploidy classes of 64N and 128N also beingdetected in these cultures (N. Debili et al., supra). In addition,megakaryocytes produced from these cultures undergo a terminalmaturation process and appear to develop proplatelets and shed plateletlike structures into the medium (F C. Zeigler et al., supra; N. Debiliet al., supra; E S. Choi et al., supra). Significantly, the plateletsproduced from such cultures have been shown to be morphologically andfunctionally indistinct from plasma-derived platelets (E S. Choi et al.,supra).

Although, rTPO appears to act directly on hematopoietic progenitors toinduce megakaryocyte differentiation, it also acts synergistically andadditively with early and late acting hematopoietic factors. In murinemegakaryocytopoiesis assays IL-11, kit ligand (KL) or EPO actsynergistically and IL-3 and IL-6 act additively with rTPO to stimulateproliferation of megakaryocyte progenitors (V C. Broudy et al., supra).In human megakaryocytopoiesis assays IL-3 and IL-6 effects are additiveto rTPO, while KL acts synergistically with rTPO (J L. Nichol et al.,supra; N. Banu et al., supra; N. Debili et al., supra; P. Angchaisuksiriet al., supra). None of the cytokines mentioned above affect themegakaryocyte maturational activity of rTPO.

The initial studies with rTPO clearly indicate that TPO predominantlyaffects the megakaryocytic lineage. However, like all otherhematopoietic regulators, TPO affects other hematopoietic lineages aswell. In the presence of EPO, rTPO has been shown to enhance erythroidburst (BFU-E) formation in human CD34+ colony assays (M. Kobayashi etal., Blood 86:2494 (1995); T. Papayannopoulou et al., Blood 87:1833(1996)). The burst promoting activity of rTPO is comparable to GM-CSFand KL and increases both the number and size of BFU-E colonies (M.Kobayashi et al., supra). In addition rTPO also stimulates CFU-Edevelopment, indicating that TPO acts on both early and late erythroidprogenitors (M. Kobayashi et al., supra; T. Papayannopoulou et al.,supra). In the absence of EPO, however, rTPO has no effect onetythropoiesis. An effect of rTPO on myeloid colony growth in normalhematopoietic cultures has not been demonstrated in vitro, however.

rTPO has a dramatic effect on platelet production when administered tonormal animals. Pharmacological doses of recombinant forms of TPO causeas much as a 10 fold increase in platelet levels in mice and non-humanprimates (E F. Winton et al., Exp. Hematol. 23:879 (1995); A M. Fareseet al., Blood 86:54 (1995); K H. Sprugel et al., Blood 86(suppl 1):20(1995); L A. Harker et al., Blood 87:1833 (1996); K. Kaushansky et al.,Exp. Hematol. 24:265 (1996); T R. Ulich et al., Blood 87:5006 (1996); K.Ault et al., Blood 86(suppl 1): 367 (1995); N C. Daw et al., Blood 86(suppl 1):5006 (1995)). This effect of rTPO is due to an increase in thesynthesis of new platelets as reticulated platelets increase within 24hours after rTPO administration (K. Ault et al., supra). Preceding thiseffect is a dramatic increase in CFU-MK in both the marrow and spleen (AM. Farese et al., supra; K. Kaushansky et al., supra; T R. Ulich et al.,supra). Megakaryocytes from rTPO treated animals exhibit a higher meanploidy and are larger in size than megakaryocytes from control animals.These later two observations again demonstrate the proliferative andmaturational activities of TPO on the megakaryocytic lineage. Becausethe effect of TPO on megakaryocytes precedes its effect on plateletproduction it has been suggested that TPO primarily affectsmegakaryocyte progenitors rather than inducing platelet release frommature megakaryocytes (N C. Daw et al., supra). No significant effect onred blood cell (RBC) or white blood cell (WBC) production occurs innormal animals following rTPO administration. However, rTPO treatmentcaused an expansion of BFU-E and CFU-GM and a redistribution CFU-E innormal mice (K. Kaushansky et al., supra) and expanded CFU-mixed inrhesus monkeys (A M. Farese et al., supra).

Even though rTPO dramatically stimulates platelet production, it onlyhas a modest effect on platelet function. In vitro studies show thatrTPO has no effect on platelet aggregation itself, but does enhanceagonist induced aggregation (G. Montrucchio et al., Blood 87:2762(1996); A. Oda et al., Blood 87:4664 (1996); C F. Toombs et al., Thromb.Res. 80:23 (1995); C F. Toombs et al., Blood 86(suppl 1):369 (1995)).rTPO appears to sensitize platelets making them moderately moreresponsive to aggregation agonist. This raises the possibility that rTPOmay have prothrombotic effects in vivo. However, an increase inthrombotic episodes in animals treated with rTPO has never beenobserved, even when platelet levels were 4-10 fold above normal. In vivothrombosis models also indicate that elevated platelet levels followingrTPO treatment is not associated with an increase in platelet dependentthrombosis (L A. Harker et al., supra, C F. Toombs et al., supra). Theseresults indicate that stimulation of platelet production by rTPO willunlikely be associated with an increase in thrombo-occulsive events.

The involvement of c-Mpl and TPO in the control of platelet productionand its effect on other hematopoietic lineages is further demonstratedby the phenotype of mice deficient in either the c-mpl or the TPO genes(W S. Alexander et al., Blood 87:2162 (1996); F J. de Sauvage et al., J.Exp. Med. 183:651 (1996); A L. Gurney et al., Science 265:1445 (1994)).In both cases a dramatic 85 to 90% drop in platelet counts is observedwith a similar decrease of megakaryocytes in the spleen and bone marrow.In addition, the megakaryocytes of the knockout mice are smaller andexhibit a lower ploidy than those of control mice. The similarity inphenotype observed for these knock-outs (KO) indicates that the systemis non-redundant and that there is probably only one receptor for TPOand one ligand for c-Mpl. Although the platelet number is reduced in theKO mice their platelets appear normal, both structurally andfunctionally, and are sufficient to prevent overt bleeding. The genesand factors involved in the production of this basal level of plateletsand megakaryocytes still remain to be identified. However, treatment ofeither the TPO or c-mpl knockout mice with other cytokines withmegakaryopoietic activity (IL-6, IL-11 and stem cell factor) results ina modest stimulation of platelet production (A L. Gurney et al., supra).This suggest that these cytokines do not require TPO or c-mpl to exerttheir thrombopoietic activity and, therefore, may be involved in themaintenance of a basal level of megakaryocytes and platelets.

Comparison of CFU-megakaryocyte (CFU-Meg) from TPO or c-mpl deficientand normal mice shows that the number of megakaryocytes progenitors isdecreased in both knock-outs compared to control, suggesting that TPOacts on very early megakaryocyte progenitors. In addition, botherythroid and myeloid progenitors are also reduced in the TPO and c-Mplknockout mice (W S. Alexander et al., supra, K. Carver-Moore et al.,88:803 (1996)). This reduction in progenitors from all lineagesindicates that TPO probably acts on a very early pluripotent progenitorcell. The involvement of TPO and c-Mpl at an early stage ofhematopoiesis correlates with the detection of c-Mpl expression inAA4+Sca+ murine stem cell population (F C. Zeigler et al., supra). Theeffect of TPO on this most primitive stem cell population still remainsto be investigated, however, preliminary data indicate that TPO maydirectly affect the proliferation of primitive murine hematopoietic stemor progenitor cells (E. Stinicka et al., Blood 87:4998 (1996); M.Kobayashi et al., Blood 88:429 (1996); H. Ku et al., Blood 87:4544(1996)). This, in part, may explain the effect TPO has on erythropoiesisand myelopoiesis in vitro and in vivo.

It has long been observed that an inverse correlation exists betweenplasma megakaryopoietic and thrombopoietic activity and platelet levels(reviewed in T P. McDonald, Am. J. Pediatr. Hematol./Oncol. 14:8(1992)). TPO specific ELISAs and cell proliferation assays have nowconfirmed that TPO levels increase and decrease inversely with plateletmass (J L. Nichol et al., supra, E V B. Emmons et al., Blood 87:4068(1996); H. Oh et al., Blood 87:4918 (1996); M. Chang et al., Blood86(suppl 1):368 (1995)). Unlike erythropoietin, however, TPO does notappear to be regulated at the transcriptional level, but rather byplatelet mass. This was initially proposed de Gabriele and Pennington(G. de Gabriele et al., Br. J. Haematol. 13:202 (1967); G. de Gabrieleet al., Br. J. Haematol. 13:210 (1967)) and subsequently confirmed byKuter and Rosenberg (D J. Kuter et al., Blood 84:1464 (1994)) who showeddirect regulation of circulating TPO levels by exogenously administeringplatelets to thrombocytopenic mice. More recently, it was demonstratedthat TPO mRNA levels in thrombocytopenic mice are not increased eventhough TPO levels are elevated by at least 10 fold (P J. Fielder et al.,Blood 87:2154 (1996); R. Stoffel et al., Blood 87:567 (1996)). Inaddition, the gene dosage effect observed in TPO heterozygous knockoutmice refute the regulation of TPO production by platelet mass (F J. deSauvage et al., supra). Taken together, these results strongly supportthe hypothesis that TPO expression is constitutive and it is thesequestering by platelets that regulates TPO levels. Platelets bind TPOwith high affinity (Kd(100-400 pM) and internalize and degrade TPO (P J.Fielder et al., supra). Platelets from c-Mpl knockout mice do not bindTPO and the clearance of TPO by these mice is S fold slower than thatobserved for wild type mice (P J. Fielder et al., supra). These resultsindicate that TPO clearance is mediated by platelet binding via c-Mpl.It is also likely that megakaryocyte mass plays a role in regulatingcirculating TPO levels. This is supported by the observation that bothITP patients and mice deficient in the NF-E2 transcription factor arehighly thrombocytopenic, exhibit megakaryocytosis, but have normal TPOlevels (E V B. Emmons et al., supra; R A. Shivdasani et al., Cell 81:695(1995)). In situ studies with radiolabeled TPO show that marrowmegakaryocytes of the NF-E2 mice bind significant amounts of labeled TPO(R A. Shivdasani et al., Blood submitted (1996)). The phenotype of theITP and NF-E2 knockout mice, therefore, suggest that binding of TPO tomegakaryocytes may also regulate TPO levels.

The dramatic effect of rTPO on platelet production in normal mice andmonkeys and subsequent clinical trials indicate that rTPO is clinicallyuseful in alleviating thrombocytopenia associated with myelosuppressiveand mycloablative therapies for cancer patients. In severalmyelosuppressive and mycloablative murine and monkey preclinical modelsrecombinant forms of TPO have been shown to significantly affectplatelet recovery. In mice treated with carboplatin and sublethalirradiation in combination (J P Leonard et al., Blood 83:1499 (1994)),daily treatment with rTPO both reduced the severity of the plateletnadir and accelerated platelet recovery by 10-12 days when compared toexcipient treated animals (G R. Thomas et al., supra; K. Kaushansky etal., supra, M M. Hokom et al., Blood 86:4486 (1995)). Similar resultswere obtained in a murine sublethal irradiation model (G R. Thomas etal., supra). In murine mycloablative transplantation models rTPO hasbeen shown to reduce the extent of the nadir and accelerate plateletrecovery by 2-3 weeks (G R. Thomas et al., supra; K. Kabaya et al.,Blood 86(suppl l):114 (1995); G. Molineux et al., Blood 86(suppl 1):227(1995)). Treatment of sublethally irradiated rhesus monkeys with rTPOaccelerated platelet recovery by 3 weeks and prevented platelet nadirsbelow 40,000 (A M. Farese et al., J. Clin. Invest. 97:2145 (1996); K J.Neelis et al., Blood 86(suppl 1):256 (1995)). Even more impressively,rTPO completely prevented post-chemotherapy thrombocytopenia followingthe treatment of rhesus monkeys with hepsulfam (A M. Farese et al.,supra). In contrast to these promising results, two groups have reportedthat rTPO had no effect on the hematopoietic recovery of lethallyirradiated mice or monkeys rescued with a marrow transplant (K J. Neeliset al, supra; W E. Fibbe et al., Blood 86:3308 (1995)). The reason forthis discrepancy is unclear, however it is possible that lethalradiation may destroy stromal cells or components essential for TPOactivity in vivo. In support of this, lethally irradiated micetransplanted with marrow cells from rTPO treated donor mice showaccelerated recovery of platelets and RBCs, however, post-transplantadministration of rTPO had no further effect on this acceleratedrecovery (W E. Fibbe et al., supra). This result suggests that althoughthe transplanted cell population was enriched for megakaryocyteprogenitors, TPO had no effect on these progenitors in a lethallyirradiated marrow.

Although rTPO only modestly affects erythroid and myeloid lineages innormal mice it dramatically accelerates the recovery of all progenitorclasses in myelosuppressed mice and monkeys resulting in a significantacceleration of RBC and WBC recovery (K. Kaushansky et al., supra; A M.Farese et al., supra; K. Kaushansky et al., J. Clin. Invest. 96:1683(1995)). The effect of rTPO on neutrophil recovery has been shown to beadditive to that of G-CSF (A M. Farese et al., supra). These resultsindicate that the clinical utility of rTPO may be broader thanoriginally anticipated.

The difference between the effect of rTPO on hematopoiesis in normal andmyelosuppressed animals is likely due to the change in the cytokineenvironment that occurs following myelosuppressive therapy. It is likelythat elevated levels of EPO, G-CSF or other cytokines essential forerythropoiesis and myelopoiesis present following myelosuppressivetreatment interact with rTPO to have a multilineage effect (K.Kaushansky et al., supra). In normal mice the level of these cytokinesare insufficient and the effects of rTPO on erythroid and myeloidlineages are less significant. This hypothesis is supported by the abovementioned synergistic interaction of rTPO and EPO to stimulate in vitroerythropoiesis (E S. Choi et al., supra). It has also been proposed thatproduction of hemopoietic factors from megakaryocytes themselves mayalso play a role in the multilineage effect of rTPO (A M. Farese et al.,supra).

In the above mentioned animal studies rTPO was administered daily for14-28 days, which was based on previous experience in dosing otherhematopoietic growth factors. However, it has recently been shown that asingle dose of rTPO following myelosuppressive treatment of mice withcarboplatin and sublethal irradiation is as effective as multiple dosesin reducing nadirs and accelerating platelet and RBC recovery (G R.Thomas et al., supra). This effect is likely due to the potency and longhalf life of rTPO.

(G R. Thomas et al., supra). This is supported by the fact that singledoses of unglycosylated rTPO153 are not effective in this model. Theseobservations indicate that the frequency of rTPO dosing required toaffect hematopoietic recovery following myelosuppressive treatment maybe significantly less than that for other currently used cytokines.

Early results from human clinical trails show that rTPO also stimulatesplatelet production in humans. In phase I trials, a pegylated andtruncated form of rTPO (MGDF) administered daily for 10 days at 0.03-5.0μg/kg to cancer patients prior to chemotherapy caused up to a four foldincrease in circulating platelet levels (R. Basser et al., Blood86(suppl 1): 257 (1995); J E J. Rasko et al., Blood 86(suppl 1):497(1995)).Similarly, patients given a single dose of rTPO had plateletlevels increase by four fold (S. Vaden-Raj et al., Stimulation ofmegakaryocyte and platelet production by a single dose of recombinanthuman thrombopoietin in cancer patients. Submitted. (1996)). In bothstudies platelet increases are observed by day four and peak about 12-16days later. No drug related toxicity's were reported and, althoughplatelet levels greater then 1×106/μl were observed in some of thepatients, no thrombotic events were observed. This indicates that TPOwill be well tolerated in humans. In myelosuppressed patients, pegylatedrTPO153(MGDF) given post chemotherapy has been shown to reduce theextent of the platelet nadir following chemotherapy (G. Begley et al.,Proceedings of ASCO 15:271 (1996); M. Fanucchi et al., Proceedings ofASCO 15:271 (1996)). As seen in the preclinical animals studies, TPOalso expanded marrow progenitors of megakaryocyte, erythroid, myeloidand multipotential lineages (S. Vaden-Raj et al., supra). This laterobservation suggests that rTPO may be useful as a priming agent.

It is believed that the proliferation and maturation of hematopoieticcells is tightly regulated by factors that positively or negativelymodulate pluripotential stem cell proliferation and multilineagedifferentiation. These effects are mediated through the high-affinitybinding of extracellular protein factors (ligands) to specific cellsurface receptors. These cell surface receptors share considerablehomology and are generally classified as members of the cytokinereceptor superfamily. Members of the superfamily include receptors for:IL-2 (b and g chains) (Hatakeyama et al., Science, 244:551-556 (1989);Takeshita et al., Science, 257:379-382 (1991)), IL-3 (Itoh et al.,Science, 247:324-328 (1990); Gorman et al., Proc. Natl. Acad. Sci. USA,87:5459-5463 (1990); Kitamura et al., Cell, 66:1165-1174 (1991a);Kitamura et al., Proc. Natl. Acad. Sci. USA, 88:5082-5086 (1991b)), IL-4(Mosley et al., Cell, 59:335-348 (1989), IL-5 (Takaki et al., EMBO J.,9:4367-4374 (1990); Tavernier et al., Cell, 66:1175-1184 (1991)), IL-6(Yamasaki et al., Science, 241:825-828 (1988); Hibi et al., Cell,63:1149-1157 (1990)), IL-7 (Goodwin et al., Cell, 60:941-951 (1990)),IL-9 (Renault et al., Proc. Natl. Acad. Sci. USA, 89:5690-5694 (1992)),granulocyte-macrophage colony-stimulating factor (GM-CSF) (Gearing etal., EMBO J., 8:3667-3676 (1991); Hayashida et al., Proc. Natl. Acad.Sci. USA, 244:9655-9659 (1990)), granulocyte colony-stimulating factor(G-CSF) (Fukunaga et al. Cell, 61:341-350 (1990a); Fukunaga et al.,Proc. Natl. Acad. Sci. USA, 87:8702-8706 (1990b); Larsen et al., J. Exp.Med, 172:1559-1570 (1990)), EPO (D'Andrea et al., Cell, 57:277-285(1989); Jones et al., Blood, 76:31-35 (1990)), Leukemia inhibitoryfactor (LIF) (Gearing et al., EMBO J., 10:2839-2848 (1991)), oncostatinM (OSM) (Rose et al., Proc. Natl. Acad. Sci. USA, 88:8641-8645 (1991))and also receptors for prolactin (Boutin et al., Proc. Natl. Acad. Sci.USA, 88:7744-7748 (1988); Edery et al., Proc. Natl. Acad. Sci. USA,86:2112-2116 (1989)), growth hormone (GH) (Leung et al., Nature,330:537-543 (1987)) and ciliary neurotrophic factor (CNTF) (Davis etal., Science, 253:59-63 (1991).

Members of the cytokine receptor superfamily may be grouped into threefunctional categories (for review see Nicola et al., Cell, 67:1-4(1991)). The first class comprises single chain receptor such aserythropoietin receptor (EPO-R) or granulocyte colony stimulating factorreceptor (G-CSF-R), which bind ligand with high affinity via theextracellular domain and also generate an intracellular signal. A secondclass of receptors, so called a-subunits, includes interleukin-6receptor (IL6-R), granulocyte-macrophage colony stimulating factorreceptor (GM-CSF-R), interleukin-3 receptor (IL3-Ra) and other membersof the cytokine receptor superfamily. These a-subunits bind ligand withlow affinity but cannot transduce an intracellular signal. A highaffinity receptor capable of signaling is generated by a heterodimerbetween an a-subunit and a member of a third class of cytokinereceptors, termed b-subunits, e.g., b_(C), the common b-subunit for thethree a-subunits of IL-3-R, IL-5-R and GM-CSF-R (Nicola N. A. et. al.Cell 67:1-4 (1991)).

Evidence that mpl is a member of the cytokine receptor superfamily comesfrom sequence homology (Gearing, EMBO J., 8:3667-3676 (1988); Bazan,Proc. Natl. Acad. Sci. USA, 87:6834-6938 (1990); Davis et al., Science,253:59-63 (1991) and Vigon et al., Proc. Natl. Acad. Sci. USA,89:5640-5644 (1992)) and its ability to transduce proliferative signals.

Deduced protein sequence from molecular cloning of murine c-mpl revealsthis protein is homologous to other cytokine receptors. Theextracellular domain contains 465 amino acid residues and is composed oftwo subdomains each with four highly conserved cysteines and aparticular motif in the N-terminal subdomain and in the C-terminalsubdomain. The ligand-binding extracellular domains are predicted tohave similar double b-barrel fold structural geometries. This duplicatedextracellular domain is highly homologous to the signal transducingchain common to IL-3, IL-5 and GM-CSF receptors as well as thelow-affinity binding domain of LIF (Vigon et al., Oncogene, 8:2607-2615(1993)). Thus mpl may belong to the low affinity ligand binding class ofcytokine receptors.

A comparison of murine mpl and mature human mpl P, reveals these twoproteins show 81% sequence identity. More specifically, the N-terminusand C-terminus extracellular subdomains share 75% and 80% sequenceidentity respectively. The most conserved mpl region is the cytoplasmicdomain showing 91% amino acid identity, with a sequence of 37 residuesnear the transmembrane domain being identical in both species.Accordingly, mpl is reported to be one of the most conserved members ofthe cytokine receptor superfamily (Vigon supra).

Activation of certain hematopoietic receptors is believed to cause oneor more effects including; stimulation of proliferation, stimulation ofdifferentiation, stimulation of growth and inhibition of apoptosis(Libol et al Proc. Natl. Acad. Sci. 248:378 (1993). Activation ofhematopoietic receptors upon ligand binding may be due to dimerizationof two or more copies of the receptor. In addition to the naturallyoccurring ligand causing this dimerization, agonist antibodies may alsoactivate receptors by crosslinking or otherwise causing dimerization ofa receptor. Such antibodies are useful for the same indications as thenatural ligand and may have advantageous properties such as a longerhalf-life. An example of a monoclonal antibody to a cytokine receptorthat activates the erythropoietin receptor (EPO-R) is described in WO96/03438 (published Feb. 8, 1996). These agonist antibodies to EPO-R areabout 3-4 orders of magnitude weaker in activity based on weight thanthe natural EPO ligand.

There is a current and continuing need to isolate and identifymolecules, especially antibodies, fragments and derivatives thereof,capable of stimulating proliferation, differentiation and maturationand/or modulation of apoptosis of cells, for example hematopoieticcells, including megakaryocytes or their predecessors for therapeuticuse in the treatment of hematopoietic disorders includingthrombocytopenia.

SUMMARY OF THE INVENTION

Accordingly, It is an object of this invention to obtain apharmaceutically or essentially pure antibody or fragments orderivatives thereof capable of stimulating proliferation,differentiation and/or maturation of hematopoietic cells, includingmegakaryocytes or their predecessors, or to modulate apoptosis ofhematopoietic cells.

It is a specific object of the present invention to isolate antibodyligands capable of binding in vivo a hematopoietic growth factorsuperfamily receptor and to activate the receptor, the antibody having abiological activity equal to or not less than 2 orders of magnitudebelow that of the naturally occurring ligand on a weight basis.

It is also an object of the present invention to isolate antibodyligands capable of binding to and activating any of the three functionalcategories of cytokine superfamily receptors (see Nicola et al., Cell,67:1-4 (1991)).

In one embodiment, the objects of the invention are achieved byproviding an antibody or fragment thereof that activates a hematopoieticgrowth factor superfamily receptor having a biological activity within 2orders of magnitude (100), preferably within one order of magnitude(10), of the natural ligand on a weight basis. Preferably, the antibodyactivates the thrombopoietin (TPO) receptor. This antibody, referred toas an agonist antibody, activates a thrombopoietin receptor whichpreferably comprises a mammalian c-mpl, more preferably human c-mpl.Usually the antibody will be a full length antibody such as an IgGantibody. Suitable presentative fragment agonist antibodies include Fv,ScFv, Fab, F(ab′)₂ fragments, as well as diabodies and linearantibodies. These fragments may be fused to other sequences including,for example, the F″ or Fc region of an antibody, a “leucine zipper” orother sequences including pegylated sequences or Fc mutants used toimprove or modulate half-life. Normally the antibody is a human antibodyand may be a non-naturally occurring antibody, including affinitymatured antibodies. Representative antibodies that activate c-mpl areselected from the group 12E10, 12B5, 1OF6 and 12D5, and affinity maturedderivatives thereof. Other preferred agonist antibodies to c-mpl areselected from the group consisting of Ab1, Ab2, Ab3, Ab4, Ab5 and Ab6,wherein each Ab1-Ab6 contains a VH and VL chain and each VH and VL chaincontains complementarity determining region (CDR) amino acid sequencesdesignated CDR1, CDR2 and CDR3 separated by framework amino acidsequences, the amino acid sequence of each CDR in each VH and VL chainof Ab1-Ab6 is shown in Table 1.

TABLE 1 Ab1: VH^(CDR1) VH^(CDR2) VH^(CDR3) DNA (SEQ ID NO: 1) (SEQ IDNO: 3) (SEQ ID NO: 5) protein (SEQ ID NO: 2) (SEQ ID NO: 4) (SEQ ID NO:6) VL^(CDR1) VL^(CDR2) VL^(CDR3) DNA (SEQ ID NO: 7) (SEQ ID NO: 9) (SEQID NO: 11) protein (SEQ ID NO: 8) (SEQ ID NO: 10) (SEQ ID NO: 12) Ab2:VH^(CDR1) VH^(CDR2) VH^(CDR3) DNA (SEQ ID NO: 13) (SEQ ID NO: 15) (SEQID NO: 17) protein (SEQ ID NO: 14) (SEQ ID NO: 16) (SEQ ID NO: 18)VL^(CDR1) VL^(CDR2) VL^(CDR3) DNA (SEQ ID NO: 19) (SEQ ID NO: 21) (SEQID NO: 23) protein (SEQ ID NO: 20) (SEQ ID NO: 22) (SEQ ID NO: 24) Ab3:VH^(CDR1) VH^(CDR2) VH^(CDR3) DNA (SEQ ID NO: 25) (SEQ ID NO: 27) (SEQID NO: 29) protein (SEQ ID NO: 26) (SEQ ID NO: 28) (SEQ ID NO: 30)VL^(CDR1) VL^(CDR2) VL^(CDR3) DNA (SEQ ID NO: 19) (SEQ ID NO: 21) (SEQID NO: 23) protein (SEQ ID NO: 20) (SEQ ID NO: 22) (SEQ ID NO: 24) Ab4:VH^(CDR1) VH^(CDR2) VH^(CDR3) DNA (SEQ ID NO: 25) (SEQ ID NO: 31) (SEQID NO: 33) protein (SEQ ID NO: 26) (SEQ ID NO: 32) (SEQ ID NO: 34)VL^(CDR1) VL^(CDR2) VL^(CDR3) DNA (SEQ ID NO: 35) (SEQ ID NO: 21) (SEQID NO: 23) protein (SEQ ID NO: 20) (SEQ ID NO: 22) (SEQ ID NO: 24) Ab5:VH^(CDR1) VH^(CDR2) VH^(CDR3) DNA (SEQ ID NO: 36) (SEQ ID NO: 38) (SEQID NO: 40) protein (SEQ ID NO: 37) (SEQ ID NO: 39) (SEQ ID NO: 41)VL^(CDR1) VL^(CDR2) VL^(CDR3) DNA (SEQ ID NO: 19) (SEQ ID NO: 21) (SEQID No: 23) protein (SEQ ID NO: 20) (SEQ ID NO: 22) (SEQ ID No: 24) Ab6:VH^(CDR1) VH^(CDR2) VH^(CDR3) DNA (SEQ ID NO: 42) (SEQ ID NO: 44) (SEQID No: 46) protein (SEQ ID NO: 43) (SEQ ID NO: 45) (SEQ ID No: 47)VL^(CDR1) VL^(CDR2) VL^(CDR3) DNA (SEQ ID NO: 48) (SEQ ID NO: 50) (SEQID No: 52) protein (SEQ ID NO: 49) (SEQ ID NO: 51) (SEQ ID No: 53)

Other preferred c-mpl agonist antibodies of this invention include thosethat activate platelets in a manner similar to TPO or in a mannersimilar to ADP, collagen and the like. Optionally the c-mpl agonistantibodies of this invention do not activate platelets. The c-mplagonist antibodies of this invention are used in a w manner similar toTPO.

In another embodiment, substantially pure single chain antibodies areprovided which bind to and act as agonist or antagonist antibodies to acytokine receptor or to a kinase receptor.

The invention also provides a method of obtaining these antibodies, inparticular a method of screening a library of phage displayedantibodies, preferably human single chain antibodies.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows examples of single chain antibody (scFv) fragmentsdenominated 10F6, 5E5, 10D10, 12B5, 12D5 and 12E10 having sequences forCDRs and framework regions.

FIG. 2 illustrates a method for the construction of a phage librarycontaining single-chain antibodies fused to a coat protein of a phage.

FIG. 3 shows a single-chain antibody displayed as a fusion protein oncoat protein 3 of a filamentous phage.

FIG. 4 illustrates a method of selecting scFv in a phage library by oneor more binding selection cycles.

FIG. 5 illustrates a method of panning high affinity phage usingbiotinylated antigen and streptavidin coated paramagnetic beads.

FIG. 6 shows a process for identifying c-mpl binding phage using a phageELISA method.

FIG. 7 illustrates DNA fingerprinting of clones to determine diversityby BstNI restriction enzyme analysis.

FIGS. 8A-C show a typical BstNI analysis on a 3% agarose gel; seeExample 2.

FIG. 9 shows the results of agonist antibodies relative to TPO in theKIRA-ELISA assay.

FIGS. 10A-F show the results of TPO-antibody competitive binding assaysfor HU-03 cells. See Example 1.

FIG. 11 shows activity for MuSK agonist antibodies of Example 5.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS I. Definitions

In general, the following words or phrases have the indicated definitionwhen used in the description, examples, and claims.

The terms “agonist” and “agonistic” when used herein refer to ordescribe a molecule which is capable of, directly or indirectly,substantially inducing, promoting or enhancing cytokine biologicalactivity or cytokine receptor activation.

“Agonist antibodies”(aAb) are antibodies or fragments thereof thatpossess the property of binding to a cytokine superfamily receptor andcausing the receptor to transduce a survival, proliferation, maturationand/or differentiation signal. Included within the definition oftransducing a survival signal is a signal which modulates cell survivalor death by apoptosis. To be therapeutically useful the agonistantibodies of this invention will be capable of inducing or causingsurvival, proliferation, maturation or differentiation at aconcentration equal to or not less than 2 orders of magnitude (100-fold)below that of the natural in vivo ligand on a weight basis.

“Activate a receptor”, as used herein, is used interchangeably withtransduce a growth, survival, proliferation, maturation and/ordifferentiation signal.

“Activate platelets”, as used herein, means to stimulate platelets tomake them more likely to aggregate by comparison to unactivatedplatelets. For example, ADP and collagen are substances known toactivate platelets.

“Affinity matured antibodies” are antibodies that have had their bindingaffinity and/or biological activity increased by altering the type orlocation of one or more residues in the variable region. An example ofalteration is a mutation which may be in either a CDR or a frameworkregion. An affinity matured antibody will typically have its bindingaffinity increased above that of the isolated or natural antibody orfragment thereof by from 2 to 500 fold. Preferred affinity maturedantibodies will have nanomolar or even picomolar affinities to thereceptor antigen. Affinity matured antibodies are produced by proceduresknown in the art. Marks, J. D. et al. Bio/Technology 10:779-783 (1992)describes affinity maturation by VH and VL domain shuffling. Randommutagenesis of CDR and/or framework residues is described by; Barbas, C.F. et al. Proc Nat. Acad. Sci, USA 91:3809-3813 (1994), Schier, R. etal. Gene 169:147-155 (1995), Yelton, D. E. et al. J. Immunol.155;1994-2004 (1995), Jackson, J. R. et al. J. Immunol. 154(7):3310-9(1995), and Hawkins, R. E. et al, J. Mol. Biol. 226:889-896 (1992).

“Cytokine” is a generic term for proteins released by one cellpopulation which act on another cell as intercellular mediators.Examples of such cytokines are lymphokines, monokines, and traditionalpolypeptide hormones. Included among the cytokines are growth hormone,insulin-like growth factors, human growth hormone, N-methionyl humangrowth hormone, bovine growth hormone, parathyroid hormone, thyroxine,insulin, proinsulin, relaxin, prorelaxin, glycoprotein hormones such asfollicle stimulating hormone (FSH), thyroid stimulating hormone (TSH),and leutinizing hormone (LH), hematopoietic growth factor, hepaticgrowth factor, fibroblast growth factor, prolactin, placental lactogen,tumor necrosis factor-a (TNF-a and TNF-b) mullerian-inhibitingsubstance, mouse gonadotropin-associated peptide, inhibin, activin,vascular endothelial growth factor, integrin, nerve growth factors suchas NGF-b, platelet-growth factor, transforming growth factors (TGFs)such as TGF-a and TGF-b, insulin-like growth factor-I and -II,erythropoietin (EPO), osteoinductive factors, interferons such asinterferon-a, -b, and -g, colony stimulating factors (CSFs) such asmacrophage-CSF (M-CSF), granulocyte-macrophage-CSF (GM-CSF), andganulocyte-CSF (G-CSF), thrombopoietin (TPO), interleukins (IL's) suchas IL-1, IL-1a, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11,IL-12 and other polypeptide factors including LIF, SCF, and kit-ligand.As used herein the foregoing terms are meant to include proteins fromnatural sources or from recombinant cell culture. Similarly, the termsare intended to include biologically active equivalents; e.g., differingin amino acid sequence by one or more amino acids or in type or extentof glycosylation.

“Cytokine superfamily receptors” and “hematopoietic growth factorsuperfamily receptors” are used interchangeably herein and are a groupof closely related glycoprotein cell surface receptors that shareconsiderable homology including frequently a WSXWS domain and aregenerally classified as members of the cytokine receptor superfamily(see e.g. Nicola et al., Cell, 67:14 (1991) and Skoda, R. C. et al. EMBOJ. 12:2645-2653 (1993)). Generally, these receptors are interleukins(IL) or colony-stimulating factors (CSF). Members of the superfamilyinclude, but are not limited to, receptors for IL-2 (b and g chains)(Hatakeyama et al., Science, 244:551-556 (1989); Takeshita et al.,Science, 257:379-382 (1991)), IL-3 (Itoh et al., Science, 247:324-328(1990); Gorman et al., Proc. Natl. Acad. Sci. USA, 87:5459-5463 (1990);Kitamura et al., Cell, 66:1165-1174 (1991a); Kitamura et al., Proc.Natl. Acad. Sci. USA, 88:5082-5086 (1991b)), IL-4 (Mosley et al., Cell,59:335-348(1989), IL-5 (Takaki et al., EMBO J., 9:4367-4374 (1990);Tavernier et al., Cell, 66:1175-1184 (1991)), IL-6 (Yamasaki et al.,Science, 241:825-828 (1988); Hibi et al., Cell, 63:1149-1157 (1990),IL-7 (Goodwin et al., Cell, 60:941-951 (1990)), IL-9 (Renault et al.,Proc. Natl. Acad. Sci. USA, 89:5690-5694 (1992)), granulocyte-macrophagecolony-stimulating factor (GM-CSF) (Gearing et al. EMBO J., 8:3667-3676(1991); Hayashida et al., Proc. Natl. Acad. Sci. USA, 244:9655-9659(1990)), granulocyte colony-stimulating factor (G-CSF) (Fukunaga et al.,Cell, 61:341-350 (1990a); Fukunaga et al., Proc. Natl. Acad. Sci. USA,87:8702-8706 (1990b); Larsen et al., J. Exp. Med, 172:1559-1570 (1990)),EPO (D'Andrea et al., Cell, 57:277-285 (1989); Jones et al., Blood,76:31-35 (1990)), Leukemia inhibitory factor (LIF) (Gearing et al., EMBOJ., 10:2839-2848 (1991)), oncostatin M (OSM) (Rose et al., Proc. Natl.Acad. Sci. USA, 88:8641-8645 (1991)) and also receptors for prolactin(Boutin e al., Proc. Natl. Acad. Sci. USA, 88:7744-7748 (1988); Edery etal., Proc. Natl. Acad. Sci. USA, 86:2112-2116 (1989)), growth hormone(GH) (Leung et al., Nature, 330:537-543 (1987)), ciliary neurotrophicfactor (CNTF) (Davis et al., Science, 253;59-63 (1991) and c-Mpl (M.Souyri et al., Cell 63:1137 (1990); I. Vigon et al., Proc. Natl. Acad.Sci. 89:5640 (1992)).

“Thrombocytopenia” in humans is defined as a platelet count below150×10⁹ per liter of blood.

“Thrombopoietic activity” is defined as biological activity thatconsists of accelerating the proliferation, differentiation and/ormaturation of megakaryocytes or megakaryocyte precursors into theplatelet producing form of these cells. This activity may be measured invarious assays including an in vivo mouse platelet rebound synthesisassay, induction of platelet cell surface antigen assay as measured byan anti-platelet immunoassay (anti-GPII_(b)III_(a)) for a human leukemiamegakaryoblastic cell line (CMK), and induction of polyploidization in amegakaryoblastic cell line (DAMI). A “thrombopoietin receptor” is amammalian polypeptide receptor which, when activated by a ligand bindingthereto, includes, causes or otherwise gives rise to “thrombopoieticactivity” in a cell or mammal, including a human.

“Control sequences” when referring to expression means DNA sequencesnecessary for the expression of an operably linked coding sequence in aparticular host organism. The control sequences that are suitable forprokaryotes, for example, include a promoter, optionally an operatorsequence, a ribosome binding site, and possibly, other as yet poorlyunderstood sequences. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

“Operably linked” when referring to nucleic acids means that the nucleicacids are placed in a functional relationship with another nucleic acidsequence. For example, DNA for a presequence or secretory leader isoperably linked to DNA for a polypeptide if it is expressed as apreprotein that participates in the secretion of the polypeptide; apromoter or enhancer is operably linked to a coding sequence if itaffects the transcription of the sequence; or a ribosome binding site isoperably linked to a coding sequence if it is positioned so as tofacilitate translation. Generally, “operably linked” means that the DNAsequences being linked are contiguous and, in the case of a secretoryleader, contiguous and in reading phase. However, enhancers do not haveto be contiguous. Linking is accomplished by ligation at convenientrestriction sites. If such sites do not exist, the syntheticoligonucleotide adapters or linkers are used in accord with conventionalpractice.

“Exogenous” when referring to an element means a nucleic acid sequencethat is foreign to the cell, or homologous to the cell but in a positionwithin the host cell nucleic acid in which the element is ordinarily notfound.

“Cell,” “cell line,” and “cell culture” are used interchangeably hereinand such designations include all progeny of a cell or cell line. Thus,for example, terms like “transformants” and “transformed cells” includethe primary subject cell and cultures derived therefrom without regardfor the number of transfers. It is also understood that all progeny maynot be precisely Identical in DNA content, due to deliberate orinadvertent mutations. Mutant progeny that have the same function orbiological activity as screened for in the originally transformed cellare included. Where distinct designations are intended, it will be clearfrom the context.

“Plasmids” are autonomously replicating circular DNA moleculespossessing independent origins of replication and are designated hereinby a lower case “p” preceded and/or followed by capital letters and/ornumbers. The stating plasmids herein are either commercially available,publicly available on an unrestricted basis, or can be constructed fromsuch available plasmids in accordance with published procedures. Inaddition, other equivalent plasmids are known in the art and will beapparent to the ordinary artisan.

“Restriction enzyme digestion” when referring to DNA means catalyticcleavage of internal phosphodiester bonds of DNA with an enzyme thatacts only at certain locations or sites in the DNA sequence. Suchenzymes are called “restriction endonucleases”. Each restrictionendonuclease recognizes a specific DNA sequence called a “restrictionsite” that exhibits two-fold symmetry. The various restriction enzymesused herein are commercially available and their reaction conditions,cofactors, and other requirements as established by the enzyme suppliersare used. Restriction enzymes commonly are designated by abbreviationscomposed of a capital letter followed by other letters representing themicroorganism from which each restriction enzyme originally was obtainedand then a number designating the particular enzyme. In general, about 1μg of plasmid or DNA fragment is used with about 1-2 units of enzyme inabout 20 μl of buffer solution. Appropriate buffers and substrateamounts for particular restriction enzymes are specified by themanufacturer. Incubation of about 1 hour at 37° C. is ordinarily used,but may vary in accordance with the supplier's instructions. Afterincubation, protein or polypeptide is removed by extraction with phenoland chloroform, and the digested nucleic acid is recovered from theaqueous fraction by precipitation with ethanol. Digestion with arestriction enzyme may be followed with bacterial alkaline phosphatasehydrolysis of the terminal 5′ phosphates to prevent the tworestriction-cleaved ends of a DNA fragment from “circularizing” orforming a closed loop that would impede insertion of another DNAfragment at the restriction site. Unless otherwise stated, digestion ofplasmids is not followed by 5′ terminal dephosphorylation. Proceduresand reagents for dephosphorylation are conventional as described insections 1.56-1.61 of Sambrook et al., Molecular Cloning: A LaboratoryManual (New York: Cold Spring Harbor Laboratory Press, 1989).

“Recovery” or “isolation” of a given fragment of DNA from a restrictiondigest means separation of the digest on polyactylamide or agarose gelby electrophoresis, identification of the fragment of interest bycomparison of its mobility versus that of marker DNA fragments of knownmolecular weight, removal of the gel section containing the desiredfragment, and separation of the gel from DNA. This procedure is knowngenerally. For example, see Lawn et al., Nucleic Acids Res., 9:6103-6114(1981), and Goeddel et al., Nucleic Acids Res., 8:4057 (1980).

“Southern analysis” or “Southern blotting” is a method by which thepresence of DNA sequences in a restriction endonuclease digest of DNA orDNA-containing composition is confirmed by hybridization to a known,labeled oligonucleotide or DNA fragment. Southern analysis typicallyinvolves electrophoretic separation of DNA digests on agarose gels,denaturation of the DNA after electrophoretic separation, and transferof the DNA to nitrocellulose, nylon, or another suitable membranesupport for analysis with a radiolabeled, biotinylated, orenzyme-labeled probe as described in sections 9.37-9.52 of Sambrook etal., supra.

“Northern analysis” or “Northern blotting” is a method used to identifyRNA sequences that hybridize to a known probe such as anoligonucleotide, DNA fragment, cDNA or fragment thereof, or RNAfragment. The probe is labeled with a radioisotope such as ³²P, or bybiotinylation, or with an enzyme. The RNA to be analyzed is usuallyelectrophoretically separated on an agarose or polyacrylamide gel,transferred to nitrocellulose, nylon, or other suitable membrane, andhybridized with the probe, using standard techniques well known in theart such as those described in sections 7.39-7.52 of Sambrook et al.,supra.

“Ligation” is the process of forming phosphodiester bonds between twonucleic acid fragments. For ligation of the two fragments, the ends ofthe fragments must be compatible with each other. In some cases, theends will be directly compatible after endonuclease digestion. However,it may be necessary first to convert the staggered ends commonlyproduced after endonuclease digestion to blunt ends to make themcompatible for ligation. For blunting the ends, the DNA is treated in asuitable buffer for at least 15 minutes at 15° C. with about 10 units ofthe Klenow fragment of DNA polymerase I or T4 DNA polymerase in thepresence of the four deoxyribonucleotide triphosphates. The DNA is thenpurified by phenol-chloroform extraction and ethanol precipitation. TheDNA fragments that are to be ligated together are put in solution inabout equimolar amounts. The solution will also contain ATP, ligasebuffer, and a ligase such as T4 DNA ligase at about 10 units per 0.5 μgof DNA. If the DNA is to be ligated into a vector, the vector is firstlinearized by digestion with the appropriate restrictionendonuclease(s). The linearized fragment is then treated with bacterialalkaline phosphatase or calf intestinal phosphatase to preventself-ligation during the ligation step.

“Preparation” of DNA from cells means isolating the plasmid DNA from aculture of the host cells. Commonly used methods for DNA preparation arethe large- and small-scale plasmid preparations described in sections1.25-1.33 of Sambrook et al., supra. After preparation of the DNA, itcan be purified by methods well known in the art such as that describedin section 1.40 of Sambrook et al., supra.

“Oligonucleotides” are short-length, single- or double-strandedpolydeoxynucleotides that are chemically synthesized by known methods(such as phosphotriester, phosphite, or phosphoramidite chemistry, usingsolid-phase techniques such as described in EP 266,032 published May 4,1988, or via deoxynucleoside H-phosphonate intermediates as described byFroehler et al., Nucl. Acids Res., 14:5399-5407 (1986)). Further methodsinclude the polymerase chain reaction defined below and other autoprimermethods and oligonucleotide syntheses on solid supports. All of thesemethods are described in Engels et al., Agnew. Chem. Int. Ed. Eng.,28:716-734 (1989). These methods are used if the entire nucleic acidsequence of the gene is known, or the sequence of the nucleic acidcomplementary to the coding strand is available. Alternatively, if thetarget amino acid sequence is known, one may infer potential nucleicacid sequences using known and preferred coding residues for each aminoacid residue. The oligonucleotides are then purified on polyacrylamidegels.

“Polymerase chain reaction” or “PCR” refers to a procedure or techniquein which minute amounts of a specific piece of nucleic acid, RNA and/orDNA, are amplified as described in U.S. Pat. No. 4,683,195 issued Jul.28, 1987. Generally, sequence information from the ends of the region ofinterest or beyond needs to be available, such that oligonucleotideprimers can be designed; these primers will be identical or similar insequence to opposite strands of the template to be amplified. The 5′terminal nucleotides of the two primers may coincide with the ends ofthe amplified material. PCR can be used to amplify specific RNAsequences, specific DNA sequences from total genomic DNA, and cDNAtranscribed from total cellular RNA, bacteriophage or plasmid sequences,etc. See generally Mullis et al., Cold Spring Harbor Symp. Quant. Biol.,51:263 (1987); Erlich, ed., PCR Technology, (Stockton Press, NY, 1989).As used herein, PCR is considered to be one, but not the only, exampleof a nucleic acid polymerase reaction method for amplifying a nucleicacid test sample comprising the use of a known nucleic acid as a primerand a nucleic acid polymerase to amplify or generate a specific piece ofnucleic acid.

“Native antibodies and immunoglobulins” are usually heterotetramericglycoproteins of about 150,000 daltons, composed of two identical light(L) chains and two identical heavy (H) chains. Each light chain islinked to a heavy chain by one covalent disulfide bond, while the numberof disulfide linkages varies between the heavy chains of differentimmunoglobulin isotypes. Each heavy and light chain also has regularlyspaced intrachain disulfide bridges. Each heavy chain has at one end avariable domain (V_(H)) followed by a number of constant domains. Eachlight chain has a variable domain at one and (V_(L)) and a constantdomain at its other end; the constant domain of the light chain isaligned with the first constant domain of the heavy chain, and the lightchain variable domain is aligned with the variable domain of the heavychain. Particular amino acid residues are believed to form an interfacebetween the light and heavy chain variable domains (Clothia et al., J.Mol. Biol., 186:651-663 (1985); Novotny and Haber, Proc. Natl. Acad.Sci. USA, 82:4592-4596 (1985)).

The term “variable” refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areused in the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not evenly distributedthrough the variable domains of antibodies. It is concentrated in threesegments called complementarity determining regions (CDRs) orhypervariable regions both in the light chain and the heavy chainvariable domains. The more highly conserved portions of variable domainsare called the framework (FR). The variable domains of native heavy andlight chains each comprise four FR regions, largely adopting a b-sheetconfiguration, connected by three CDRs, which form loops connecting, andin some cases forming part of, the b-sheet structure. The CDRs in eachchain are held together in close proximity by the FR regions and, withthe CDRs from the other chain, contribute to the formation of theantigen binding site of antibodies (see Kabat et al., Sequences ofProteins of Immunological Interest, National Institute of Health,Bethesda, Md. (1987)). The constant domains are not involved directly inbinding an antibody to an antigen, but exhibit various effectorfunctions, such as participation of the antibody in antibody-dependentcellular toxicity.

Papain digestion of antibodies products two identical antigen bindingfragments, called “Fab” fragments, each with a single antigen bindingsite, and a residual “Fc” fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields an F(ab′)₂ fragment thathas two antigen combining sites and is still capable of cross-linkingantigen.

“Fv” is the minimum antibody fragment which contains a complete antigenrecognition and binding site. This region consists of a dimer of oneheavy and one light chain variable domain in tight, non-covalentassociation. It is in this configuration that the three CDRs of eachvariable domain interact to define an antigen binding site on thesurface of the V_(H)-V_(L) dimer. Collectively, the six CDRs conferantigen binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chainand the first constant domain (CH1) of the heavy chain. Fab″ fragmentsdiffer from Fab fragments by the addition of a few residues at thecarboxy terminus of the heavy chain CH1 domain including one or morecysteines from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear a free thiol group. F(ab′)₂ antibody fragments originally wereproduced as pairs of Fab′ fragments which have hinge cysteines betweenthem. Other, chemical couplings of antibody fragments are also known.

The “light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa and lambda (1), based on the amino acid sequences of theirconstant domains.

Depending on the amino acid sequence of the constant domain of theirheavy chains, immunoglobulins can be assigned to different classes.There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG andIgM, and several of these may be further divided into subclasses(isotypes), e.g., IgG-1, IgG-2, IgG-3, and IgG-4; IgA-1 and IgA-2. Theheavy chain constant domains that correspond to the different classes ofimmunoglobulins are called alpha, delta, epsilon, gamma and μ,respectively. The subunit structures and three-dimensionalconfigurations of different classes of immunoglobulins are well known.

The term “antibody” is used in the broadest sense and specificallycovers single monoclonal antibodies (including agonist and antagonistantibodies), antibody compositions with polyepitopic specificity, aswell as antibody fragments (e.g., Fab, F(ab′)₂, scFv and Fv), so long asthey exhibit the desired biological activity.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional (polyclonal) antibody preparations which typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen. In addition to their specificity, the monoclonal antibodies areadvantageous in that they are synthesized by the hybridoma culture,uncontaminated by other immunoglobulins. The modifier “monoclonal”indicates the character of the antibody as being obtained from asubstantially homogeneous population of antibodies, and is not to beconstrued as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies to be used in accordancewith the present invention may be made by the hybridoma method firstdescribed by Kohler & Milstein, Nature, 256:495 (1975), or may be madeby recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567 (Cabillyet al.)).

The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity, e.g. binding to andactivating mpl (U.S. Pat. No. 4,816,567 (Cabilly et al.); and Morrisonet al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).

“Humanized” forms of non-human (e.g., murine) antibodies are chimericimmunoglobulins, immunoglobulin chains or fragments thereof (such as Fv,Fab, Fab′, F(ab′)₂ or other antigen-binding subsequences of antibodies)which contain minimal sequence derived from non-human immunoglobulin.For the most part, humanized antibodies are human immunoglobulins(recipient antibody) in which residues from a complementary determiningregion (CDR) of the recipient are replaced by residues from a CDR of anon-human species (donor antibody) such as mouse, rat or rabbit havingthe desired specificity, affinity and capacity. In some instances, Fvframework residues of the human immunoglobulin are replaced bycorresponding non-human residues. Furthermore, humanized antibody maycomprise residues which are found neither in the recipient antibody norin the imported CDR or framework sequences. These modifications are madeto further refine and optimize antibody performance. In general, thehumanized antibody will comprise substantially all of at least one, andtypically two, variable domains in which all or substantially all of theCDR regions correspond to those of a non-human immunoglobulin and all orsubstantially all of the FR regions are those of a human immunoglobulinconsensus sequence. The humanized antibody optimally also will compriseat least a portion of an immunoglobulin constant region (Fc), typicallythat of a human immunoglobulin. For further details see: Jones et al.,Nature, 321:522-525 (1986); Reichmann et al., Nature, 332:323-329(1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)).

“Single-chain Fv” or “sFv” antibody fragments comprise the V_(H) andV_(L) domains of antibody, wherein these domains are present in a singlepolypeptide chain. Generally, the Fv polypeptide further comprises apolypeptide linker between the V_(H) and V_(L) domains which enables thesFv to form the desired structure for antigen binding. For a review ofsFv see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315(1994).

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy chain variabledomain (V_(H)) connected to a light chain variable domain (V_(L)) in thesame polypeptide chain (V_(H) and V_(L)). By using a linker that is tooshort to allow pairing between the two domains on the same chain, thedomains are forced to pair with the complementary domains of anotherchain and create two antigen-binding sites. Diabodies are described morefully in, for example, EP 404,097; WO 93/11161; and Hollinger et al.,Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993).

The expression “linear antibodies” when used throughout this applicationrefers to the antibodies described in Zapata et al. Protein Eng. 8(10):1057-1062 (1995). Briefly, these antibodies comprise a pair of tandem Fdsegments (V_(H)-C_(H)1-V_(H)-C_(H)1) which form a pair of antigenbinding regions. Linear antibodies can be bispecific or monospecific.

A “variant” antibody, refers herein to a molecule which differs in aminoacid sequence from a “parent” antibody amino acid sequence by virtue ofaddition, deletion and/or substitution of one or more amino acidresidue(s) in the parent antibody sequence. In the preferred embodiment,the variant comprises one or more amino acid substitution(s) in one ormore hypervariable region(s) of the parent antibody. For example, thevariant may comprise at least one, e.g. from about one to about ten, andpreferably from about two to about five, substitutions in one or morehypervariable regions of the parent antibody. Ordinarily, the variantwill have an amino acid sequence having at least 75% amino acid sequenceidentity with the parent antibody heavy or light chain variable domainsequences, more preferably at least 80%, more preferably at least 85%,more preferably at least 90%, and most preferably at least 95%. Identityor homology with respect to this sequence is defined herein as thepercentage of amino acid residues in the candidate sequence that areidentical with the parent antibody residues, after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity. See FIG. 1. None of N-terminal, C-terminal,or internal extensions, deletions, or insertions into the antibodysequence shall be construed as affecting sequence identity or homology.The variant retains the ability to bind the receptor and preferably hasproperties which are superior to those of the parent antibody. Forexample, the variant may have a stronger binding affinity, enhancedability to activate the receptor, etc. To analyze such properties, oneshould compare a Fab form of the variant to a Fab form of the parentantibody or a full length form of the variant to a full length form ofthe parent antibody, for example, since it has been found that theformat of the antibody impacts its activity in the biological activityassays disclosed herein. The variant antibody of particular interestherein is one which displays at least about 10 fold, preferably at leastabout 20 fold, and most preferably at least about 50 fold, enhancementin biological activity when compared to the parent antibody.

The “parent” antibody herein is one which is encoded by an amino acidsequence used for the preparation of the variant. Preferably, the parentantibody has a human framework region and has human antibody constantregion(s). For example, the parent antibody may be a humanized or humanantibody.

An “isolated” antibody is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In preferred embodiments, the antibody will bepurified (1) to greater than 95% by weight of antibody as determined bythe Lowry method, and most preferably more than 99% by weight, (2) to adegree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, or (3)to homogeneity by SDS-PAGE under reducing or nonreducing conditionsusing Coomassie blue or, preferably, silver stain. Isolated antibodyincludes the antibody in situ within recombinant cells since at leastone component of the antibody's natural environment will not be presentOrdinarily, however, isolated antibody will be prepared by at least onepurification step.

The term “epitope tagged” when used herein refers to an antibody fusedto an “epitope tag”. The epitope tag polypeptide has enough residues toprovide an epitope against which an antibody thereagainst can be made,yet is short enough such that it does not interfere with activity of theantibody. The epitope tag preferably is sufficiently unique so that theantibody thereagainst does not substantially cross-react with otherepitopes. Suitable tag polypeptides generally have at least 6 amino acidresidues and usually between about 8-50 amino acid residues (preferablybetween about 9-30 residues). Examples include the flu HA tagpolypeptide and its antibody 12CA5 (Field et al. Mol. Cell. Biol.8:2159-2165 (1988)); the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and9E10 antibodies thereto (Evan et al., Mol. Cell. Biol. 5(12):3610-3616(1985)); and the Herpes Simplex virus glycoprotein D (gD) tag and itsantibody (Paborsky et al., Protein Engineering 3(6):547-553 (1990)). Incertain embodiments, the epitope tag is a “salvage receptor bindingepitope”. As used herein, the term “salvage receptor binding epitope”refers to an epitope of the Fc region of an IgG molecule (e.g., IgG₁.IgG₂, IgG₃, or IgG₄) that is responsible for increasing the in vivoserum half-life of the IgG molecule.

The terms “mpl ligand”, mpl ligand polypeptide”, “ML”, “thrombopoietin”or “TPO” are used interchangeably herein and include any polypeptidethat possesses the property of binding to mpl, a member of the cytokinereceptor superfamily, and having a biological property of mpl ligand. Anexemplary biological property is the ability to stimulate theincorporation of labeled nucleotides (e.g. ³H-thymidine) into the DNA ofIL-3 dependent Ba/F3 cells transfected with human mpl. Another exemplarybiological property is the ability to stimulate the incorporation of ³⁵Sinto circulating platelets in a mouse platelet rebound assay. Thisdefinition encompasses a polypeptide isolated from a mpl ligand sourcesuch as aplastic porcine plasma described herein or from another source,such as another animal species, including humans, or prepared byrecombinant or synthetic methods. Examples include TPO(332) andrhTPO₃₃₂. Also included in this definition is the thrombopoietic liganddescribed in WO 95/28907 having a molecular weight of about 31,000daltons (31 kd) as determined by SDS gel under reducing conditions and28,000 daltons (28 kd) under non-reducing conditions. The term “TPO”includes variant forms, such as fragments, alleles, isoforms, analogues,chimera thereof and mixtures of these forms. For convenience, all ofthese ligands will be referred to below simply as “TPO” recognizing thatall individual ligands and ligand mixtures are referred to by this term.

Preferably, the TPO is a compound having thrombopoietic activity orbeing capable of increasing serum platelet counts in a mammal. The TPOis preferably capable of increasing endogenous platelet counts by atleast 10%, more preferably by 50%, and most preferably capable ofelevating platelet counts in a human to greater than about 150×10⁹ perliter of blood.

The TPO of this invention preferably has at least 70% overall sequenceidentity with the amino acid sequence of the highly purifiedsubstantially homogeneous porcine mpl ligand polypeptide and at least80% sequence identity with the “EPO-domain” of the porcine mpl ligandpolypeptide. Alternatively, the TPO of this invention may be a maturehuman mpl ligand (hML), or a variant or post-transcriptionally modifiedform thereof or a protein having about 80% sequence identity with maturehuman mpl ligand. Alternatively, the TPO may be a fragment, especiallyan amino-terminus or “EPO-domain” fragment, of the mature human mplligand. Preferably, the amino terminus fragment retains substantiallyall of the human ML sequence between the first and fourth cysteineresidues but may contain substantial additions, deletions orsubstitutions outside that region. According to this embodiment, thefragment polypeptide may be represented by the formula:

X-hTPO(7-151)-Y

Where hTPO(7-151) represents the human TPO (hML) amino acid sequencefrom Cys⁷ through Cys¹⁵¹ inclusive; X represents the amino group of Cys⁷or one or more of the amino-terminus amino acid w residue(s) of themature TPO or amino acid residue extensions thereto such as Met, Lys,Tyr or amino acid substitutions thereof such as arginine to lysine orleader sequences containing, for example, proteolytic cleavage sites(e.g. Factor Xa or thrombin); and Y represents the carboxy terminalgroup of Cys¹⁵¹ or one or more carboxy-terminus amino acid residue(s) ofthe mature TPO or extensions thereto.

A “TPO fragment” means a portion of a naturally occurring mature fulllength mpl ligand or TPO sequence having one or more amino acid residuesor carbohydrate units deleted. The deleted amino acid residue(s) mayoccur anywhere in the peptide including at either the N-terminal orC-terminal end or internally, so long as the fragment shares at leastone biological property in common with mpl ligand. Mpl ligand fragmentstypically will have a consecutive sequence of at least 10, 15, 20, 25,30 or 40 amino acid residues that are identical to the sequences of thempl ligand isolated from a mammal including the ligand isolated fromaplastic porcine plasma or the human or murine ligand, especially theEPO-domain thereof. Representative examples of N-terminal fragments areTPO(153), hML₁₅₃ or TPO(Met⁻¹ 1-153).

The terms “TPO isoform(s)” and “TPO sequence isoform(s)” or the term“derivatives” in association with TPO, etc. as used herein means abiologically active material as defined below having less than 100%sequence identity with the TPO isolated from recombinant cell culture,aplastic porcine plasma or the human mpl ligand. Ordinarily, abiologically active mpl ligand or TPO isoform will have an amino acidsequence having at least about 70% amino acid sequence identity with thempl ligand/TPO isolated from aplastic porcine plasma or the maturemurine, human mpl ligand or fragments thereof, preferably at least about75%, more preferably at least about 80%, still more preferably at leastabout 85%, even more preferably at least about 90%, and most preferablyat least about 95%.

TPO “analogues” include covalent modification of TPO or mpl ligand bylinking the TPO polypeptide to one of a variety of nonproteinaceouspolymers, e.g. polyethylene glycol, polypropylene glycol, orpolyoxyalkylenes, in the manner set forth in U.S. Pat. Nos. 4,640,835;4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337. TPOpolypeptides covalently linked to the forgoing polymers are referred toherein as pegylated TPO.

Still other preferred TPO polypeptides of this invention include mplligand sequence variants and chimeras. Ordinarily, preferred mpl ligandsequence variants and chimeras are biologically active mpl ligandvariants that have an amino acid sequence having at least 90% amino acidsequence identity with the human mpl ligand and most preferably at least95%. An exemplary preferred mpl ligand variant is a N-terminal domainhML variant (referred to as the “EPO-domain” because of its sequencehomology to erythropoietin). The preferred hML EPO-domain comprisesabout the first 153 amino acid residues of mature hML and is referred toas hML₁₅₃. An optionally preferred hML sequence variant comprises one inwhich one or more of the basic or dibasic amino acid residue(s) in theC-terminal domain is substituted with a non-basic amino acid residue(s)(e.g., hydrophobic, neutral, acidic, aromatic, Gly, Pro and the like). Apreferred hML C-terminal domain sequence variant comprises one in whichArg residues 153 and 154 are replaced with Ala residues. This variant isreferred to as hML₃₃₂(R153A, R154A).

A preferred chimera is a fusion between mpl ligand or fragment (definedbelow) thereof with a heterologous polypeptide or fragment thereof. Forexample, hML₁₅₃ may be fused to an IgG fragment to improve serumhalf-life or to IL-3, G-CSF or EPO to produce a molecule with enhancedthrombopoietic or chimeric hematopoietic activity.

Other preferred mpl ligand fragments have a Met preceding the aminoterminus Ser (e.g. Met⁻¹TPO₁₅₃). This is preferred when, for example,the protein is expressed directly in a microorganism such as E. coli.Optionally, these mpl ligand fragments may contain amino acidsubstitutions to facilitate derivitization. For example, Arg₁₅₃ or otherresidues of the carbohydrate domain may be substituted with Lys tocreate additional sites to add polyethylene glycol. Preferred mpl ligandfragments according to this option include Met⁻¹TPO(1-X) where X isabout 153, 164, 191, 199, 205, 207, 217, 229, or 245 for the sequence ofresidues 1-X. Other preferred mpl ligand fragments include thoseproduced as a result of chemical or enzymatic hydrolysis or digestion ofthe purified ligand.

“Essentially pure” protein means a composition purified to removecontaminating proteins and other cellular components, preferablycomprising at least about 90% by weight of the protein, based on totalweight of the composition, more preferably at least about 95% by weight.“Essentially homogeneous” protein means a composition comprising atleast about 99% by weight of protein, based on total weight of thecomposition.

II. Preferred Embodiments of the Invention

In one embodiment, preferred antibodies of this invention aresubstantially homogeneous antibodies and variants thereof, referred toas agonist antibodies (aAb), that possess the property of binding toc-mpl, a member of the hematopoietic growth factor receptor superfamily,and transducing a survival, proliferation, maturation and/ordifferentiation signal. Such signal transduction may be determined bymeasuring stimulation of incorporation of labeled nucleotides(³H-thymidine) into the DNA of IL-3 dependent Ba/F3 cells transfectedwith human mpl P, or with a CMK Assay measuring Induction of theplatelet antigen GPII_(b)III_(a) expression. Signal transduction mayalso be determined by KIRA ELISA by measuring phosphorylation of thec-mpl-Rse.gD chimeric receptor, in a c-mpl/Mab HU-03 cell proliferationassay or in a liquid suspension megakaryocytopoiesis assay.

Preferred c-mpl agonist antibodies of this invention are also capable ofinducing or causing survival, proliferation, maturation ordifferentiation of CD34+ cells into the platelet producing form at aconcentration equal to or not less than 2 orders of magnitude (100-fold)below that of thrombopoietin on a weight basis.

More preferred c-mpl aAb(s) are substantially purified aAb(s) havinghematopoietic, especially megakaryocytopoietic or thrombocytopoieticactivity—namely, being capable of stimulating proliferation, maturationand/or differentiation of immature megakaryocytes or their predecessorsinto the mature platelet-producing form that demonstrate a biologicalactivity equal to or within 2 orders of magnitude of that of rhTPO on aweight basis. Most preferred aAb(s) of this invention are human aAb(s)including full length antibodies having an intact human Fc region andincluding fragments thereof having hematopoietic, megakaryocytopoieticor thrombopoietic activity. Exemplary fragments having the abovedescribed biological activity include; Fv, scFv, F(ab′), F(ab′)₂.

Preferred scFv fragments denominated 10F6, 5E5, 10D10, 12B5, 12D5 and12E10 having sequences for CDRs and Framework regions provided in FIG.1. Alternatively, the above enumerated scFvs are affinity matured bymutating 1-3 amino acid residues in one or more of the CDRs or in theframework regions between the CDRs.

The framework regions may be derived from a “consensus sequence” (i.e.the most common amino acids of a class, subclass or subgroup of heavy orlight chains of human immunoglobulins) or may be derived from anindividual human antibody framework region or from a combination ofdifferent framework region sequences. Many human antibody frameworkregion sequences are compiled in Kabat et al., Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991), pages 647-669), for example.

A suitable method for purifying mpl antibodies comprises contacting anantibody source containing the mpl antibody molecules with animmobilized receptor polypeptide, specifically mpl or a mpl fusionpolypeptide, under conditions whereby the mpl antibody molecules to bepurified are selectively adsorbed onto the immobilized receptorpolypeptide, washing the immobilized support to remove non-adsorbedmaterial, and eluting the molecules to be purified from the immobilizedreceptor polypeptide with an elution buffer. The source containing thempl antibody may be a library of antibodies having different bindingepitopes and the receptor may be immobilized on a plate, tube, particleor other suitable surface using known methods.

Alternatively, the source containing the antibody is recombinant cellculture where the concentration of antibody in either the culture mediumor in cell lysates is generally higher than in plasma or other naturalsources. The preferred purification method to provide substantiallyhomogeneous antibody comprises: removing particulate debris, either hostcells or lysed fragments by, for example, centrifugation orultrafiltration; optionally, protein may be concentrated with acommercially available protein concentration filter, followed byseparating the antibody from other impurities by one or more stepsselected from; immunoaffinity, ion-exchange (e.g., DEAE or matricescontaining carboxymethyl or sulfopropyl groups), Blue-SEPHAROSE, CMBlue-SEPHAROSE, MONO-Q, MONO-S, lentil lectin-SEPHAROSE, WGA-SEPHAROSE,Con A-SEPHAROSE, Ether TOYPEARL, Butyl TOYPEARL, Phenyl TOYPEARL,protein A SEPHAROSE, SDS-PAGE, reverse phase HPLC (e.g., silica gel withappended aliphatic groups) or SEPHADEX molecular sieve or size exclusionchromatography, and ethanol or anmmonium sulfate precipitation. Aprotease inhibitor such as methylsulfonylfluoride (PMSF) may be includedin any of the foregoing steps to inhibit proteolysis.

Preferably, the isolated antibody is monoclonal (Kohler and Milstein,Nature, 256:495-497 (1975); Campbell, Laboratory Techniques inBiochemistry and Molecular Biology, Burdon et al., Eds, Volume 13,Elsevier Science Publisrers, Amsterdam (1985); and Huse et al., Science,246:1275-1281 (1989)). A preferred mpl antibody is one that binds to mplreceptor with an affinity of at least about 10⁶ l/mole . More preferablythe antibody binds with an affinity of at least about 10⁷ l/mole or evenat least 10⁹ l/mole. Most preferably, the antibody is raised against ampl receptor having one of the above described effector functions. Theisolated antibody capable of binding to the mpl receptor may optionallybe fused to a second polypeptide and the antibody or fusion thereof maybe used to isolate and purify mpl from a source as described above forimmobilized mpl polypeptide. In a further preferred aspect of thisembodiment, the invention provides a method for detecting the mpl ligandin vitro or in vivo comprising contacting the antibody with a sample,especially a serum sample, suspected of containing the ligand anddetecting if binding has occurred.

The invention also provides an isolated nucleic acid molecule encodingthe mpl antibody or fragments thereof, which nucleic acid molecule maybe labeled or unlabeled with a detectable moiety, and a nucleic acidmolecule having a sequence that is complementary to, or hybridizes understringent or moderately stringent conditions with, a nucleic acidmolecule having a sequence encoding a mpl antibody. A preferred mplantibody nucleic acid is RNA or DNA that encodes a biologically activehuman antibody.

In a further preferred embodiment of this invention, the nucleic acidmolecule is cDNA encoding the mpl antibody and further comprises areplicable vector in which the cDNA is operably linked to controlsequences recognized by a host transformed with the vector. This aspectfurther includes host cells transformed with the vector and a method ofusing the cDNA to effect production of antibody, comprising expressingthe cDNA encoding the antibody in a culture of the transformed hostcells and recovering the antibody from the host cell culture. Theantibody prepared in this manner is preferably substantially homogeneoushuman antibody. A preferred host cell for producing the antibody isChinese hamster ovary (CHO) cells. An alternative preferred host cell isE. coli.

The invention further includes a preferred method for treating a mammalhaving an immunological or hematopoietic disorder, especiallythrombocytopenia comprising administering a therapeutically effectiveamount of a mpl agonist or antagonist antibody to the mammal.Optionally, the antibody is administered in combination with a cytokine,especially a colony stimulating factor or interleukin. Preferred colonystimulating factors or interleukins include; kit-ligand, LIF, G-CSF,GM-CSF, M-CSF, EPO, IL-1, IL,2, IL-3, IL-5, IL-6, IL-7, IL-8, IL-9 orIL-11. Alternatively, the antibody is administered in combination withan Insulin-like growth factor (e.g., IGF-1) or a tumor necrosis factor(e.g., lymphotoxin (LT)).

III. Methods of Making

Nucleic acid encoding the agonist and/or antagonist antibodies of theinvention can be prepared from a library of single chain antibodiesdisplayed on a bacteriophage. The preparation of such a library is wellknown to one of skill in this art. Suitable libraries may be prepared bythe methods described in WO 92/01047, WO 92/20791, WO 93106213, WO93/11236, WO 93/19172, WO 95/01438 and WO 95/15388. In a preferredembodiment, a library of single chain antibodies (scFv) may be generatedfrom a diverse population of human B-cells from human donors. mRNAcorresponding to the VH and VL antibody chains is isolated and purifiedusing standard techniques and reverse transcribed to generate apopulation of cDNA. After PCR amplification, DNA coding for single chainantibodies is assembled using a linker, such as Gly₄Ser, and cloned intosuitable expression vectors. A phage library is then prepared in whichthe population of single chain antibodies is displayed on the surface ofthe phage. Suitable methods for preparing phage libraries have beenreviewed and are described in Winter et. al., Annu. Rev. Immunol., 1994,12:433-55; Soderlind et. al., Immunological Reviews, 1992, 130:109-123;Hoogenboom, Tibtech February 1997, Vol. 15; Neri et. al., CellBiophysics, 1995, 27:47-61, and the references described therein.

The antibodies of the invention having agonist or antagonist propertiesmay be selected by immobilizing a receptor and then panning a library ofhuman scFv prepared as described above using the immobilized receptor tobind antibody. Griffiths et. al., EMBO-J, 1993, 12:725-734. Thespecificity and activity of specific clones can be assessed using knownassays. Griffiths et. al,; Clarkson et. al., Nature, 1991, 352:642-648.After a first panning step, one obtains a library of phage containing aplurality of different single chain antibodies displayed on phage havingimproved binding to the receptor. Subsequent panning steps provideadditional libraries with higher binding affinities. When avidityeffects are a problem, monovalent phage display libraries may be used inwhich less than 20%, preferably less than 10%, and more preferably lessthan 1% of the phage display more than one copy of an antibody on thesurface of the phage. Monovalent display can be accomplished with theuse of phagemid and helper phage as described, for example, in Lowmanet. al., Methods: A Companion to Methods in Enzymology, 1991,3(3):205-216. A preferred phage is M13 and display is preferably as afusion protein with coat protein 3 as described in Lowman et. al.,supra. Other suitable phage include fl and fd filamentous phage. Fusionprotein display with other virus coat proteins is also known and may beused in this invention. See U.S. Pat. No. 5,223,409.

Amino acid sequence variants of the antibody are prepared by introducingappropriate nucleotide changes into the antibody DNA, or by peptidesynthesis. Such variants include, for example, deletions from, and/orinsertions into and/or substitutions of, residues within the amino acidsequences of the antibodies of the examples herein. Any combination ofdeletion, insertion, and substitution is made to arrive at the finalconstruct, provided that the final construct possesses the desiredcharacteristics. The amino acid changes also may alterpost-translational processes of the humanized or variant antibody, suchas changing the number or position of glycosylation sites.

A useful method for identification of certain residues or regions of theantibody that are preferred locations for mutagenesis is called “alaninescanning mutagenesis,” as described by Cunningham and Wells Science,244:1081-1085 (1989). Here, a residue or group of target residues areidentified (e.g., charged residues such as arg, asp, his, lys, and glu)and replaced by a neutral or negatively charged amino acid (mostpreferably alanine or polyalanine) to affect the interaction of theamino acids with the receptor. Those amino acid locations demonstratingfunctional sensitivity to the substitutions then are refined byintroducing further or other variants at, or for, the sites ofsubstitution. Thus, while the site for introducing an amino acidsequence variation is predetermined, the nature of the mutation per seneed not be predetermined. For example, to analyze the performance of amutation at a given site, ala scanning or random mutagenesis isconducted at the target codon or region and the expressed antibodyvariants are screened for the desired activity.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue or the antibody fusedto an epitope tag. Other insertional variants of the antibody moleculeinclude the fusion to the N- or C-terminus of the antibody of an enzymeor a polypeptide which increases the serum half-life of the antibody.

Another type of variant is an amino acid substitution variant. Thesevariants have at least one amino acid residue in the antibody moleculeremoved and a different residue inserted in its place. The sites ofgreatest interest for substitutional mutagenesis include thehypervariable regions, but FR alterations are also contemplated.Conservative substitutions are shown in Table 2 under the heading of“preferred substitutions”. If such substitutions result in a change inbiological activity, then more substantial changes, denominated“exemplary substitutions” in Table 2, or as further described below inreference to amino acid classes, may be introduced and the productsscreened.

TABLE 2 Original Exemplary Preferred Residue Substitutions SubstitutionsAla (A) val; leu; ile val Arg (R) lys; gln; asn lys Asn (N) gln; his;asp, lys; arg gln Asp (D) glu; asn glu Cys (C) ser; ala ser Gln (Q) asn;glu asn Glu (E) asp; gln asp Gly (G) ala ala His (H) asn; gln; lys; argarg Ile (I) leu; val; met; ala; phe; leu norleucine Leu (L) norleucine;ile; val; met; ile ala; phe Lys (K) arg; gln; asn arg Met (M) leu; phe;ile leu Phe (F) leu; val; ile; ala; tyr tyr Pro (P) ala ala Ser (S) thrthr Thr (T) ser ser Trp (W) tyr; phe tyr Tyr (Y) trp; phe; thr; ser pheVal (V) ile; leu; mel; phe; ala; leu norleucine

Substantial modifications in the biological properties of the antibodyare accomplished by selecting substitutions that differ significantly intheir effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain. Naturallyoccurring residues are divided into groups based on common side-chainproperties:

(1) hydrophobic: norleucine, met, ala, val, leu, ile;

(2) neutral hydrophilic: cys, ser, thr;

(3) acidic: asp, glu;

(4) basic: asn, gin, his, lys, arg;

(5) residues that influence chain orientation: gly, pro; and

(6) aromatic: trp, tyr, phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

Any cysteine residue not involved in maintaining the proper conformationof the humanized or variant antibody also may be substituted, generallywith serine, to improve the oxidative stability of the molecule andprevent aberrant crosslinking. Conversely, cysteine bond(s) may be addedto the antibody to improve its stability (particularly where theantibody is an antibody fragment such as an Fv fragment).

A particularly preferred type of substitutional variant involvessubstituting one or more hypervariable region residues of a parentantibody (e.g. a humanized or human antibody). Generally, the resultingvariant(s) selected for further development will have improvedbiological properties relative to the parent antibody from which theyare generated. A convenient way for generating such substitutionalvariants is affinity maturation using phage using methods known in theart. Briefly, several hypervariable region sites (e.g. 3-7 sites) aremutated to generate all possible amino substitutions at each site. Theantibody variants thus generated are displayed in a monovalent fashionfrom filamentous phage particles as fusions to the gene III product ofM13 packaged within each particle. The phage-displayed variants are thenscreened for their biological activity (e.g. binding affinity) as hereindisclosed. In order to identify candidate hypervariable region sites formodification, alanine scanning mutagenesis can be performed toidentified hypervariable region residues contributing significantly toantigen binding. Alternatively, or in addition, it may be beneficial toanalyze a crystal structure of the antigen-antibody complex to identifycontact points between the antibody and receptor. Such contact residuesand neighboring residues are candidates for substitution according tothe techniques elaborated herein. Once such variants are generated, thepanel of variants is subjected to screening as described herein andantibodies with superior properties in one or more relevant assays maybe selected for further development.

Another type of amino acid variant of the antibody alters the originalglycosylation pattern of the antibody. By altering is meant deleting oneor more carbohydrate moieties found in the antibody, and/or adding oneor more glycosylation sites that are not present in the antibody.

Glycosylation of antibodies is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to the antibody is convenientlyaccomplished by altering the amino acid sequence such that it containsone or more of the above-described tripeptide sequences (for N-linkedglycosylation sites). The alteration may also be made by the additionof, or substitution by, one or more serine or threonine residues to thesequence of the original antibody (for O-linked glycosylation sites).

Nucleic acid molecules encoding amino acid sequence variants of theantibody are prepared by a variety of methods known in the art. Thesemethods include, but are not limited to, isolation from a natural source(in the case of naturally occurring amino acid sequence variants) orpreparation by oligonucleotide-mediated (or site-directed) mutagenesis,PCR mutagenesis, and cassette mutagenesis of an earlier prepared variantor a non-variant version of the antibody.

Preferably, the antibodies are prepared by standard recombinantprocedures which involve production of the antibodies by culturing cellstransfected to express antibody nucleic acid (typically by transformingthe cells with an expression vector) and recovering the antibody fromthe cells of cell culture.

The nucleic acid (e.g., cDNA or genomic DNA) encoding mpl antibodyselected as described above is inserted into a replicable vector forfurther cloning (amplification of the DNA) or for expression. Manyvectors are available, and selection of the appropriate vector willdepend on (1) whether it is to be used for DNA amplification or for DNAexpression, (2) the size of the nucleic acid to be inserted into thevector, and (3) the host cell to be transformed with the vector. Eachvector contains various components depending on its function(amplification of DNA or expression of DNA) and the host cell with whichit is compatible. The vector components generally include, but are notlimited to, one or more of the following: a signal sequence, an originof replication, one or more marker genes, an enhancer element, apromoter, and a transcription termination sequence.

(i) Signal Sequence Component

The mpl antibody of this invention may be expressed not only directly,but also as a fusion with a heterologous polypeptide, preferably asignal sequence or other polypeptide having a specific cleavage site atthe N-terminus of the mature protein or polypeptide. In general, thesignal sequence may be a component of the vector, or it may be a part ofthe mpl antibody DNA that is inserted into the vector. The heterologoussignal sequence selected should be one that is recognized and processed(i.e., cleaved by a signal peptidase) by the host cell. For prokaryotichost cells a prokaryotic signal sequence selected, for example, from thegroup of the alkaline phosphatase, penicillinase, lpp, or heat-stableenterotoxin II leaders. For yeast secretion the native signal sequencemay be substituted by, e.g., the yeast invertase, alpha factor, or acidphosphate leaders, the C. albicans glucoamylase leader (EP 362,179published Apr. 4, 1990), or the signal described in WO 90/13646published Nov. 15, 1990. In mammalian cell expression the native signalsequence (ie., the mpl ligand presequence that normally directssecretion of mpl ligand from its native mammalian cells in vivo) issatisfactory, although other mammalian signal sequences may be suitable,such as signal sequences from other mpl ligand polypeptides or from thesame mpl ligand from a different animal species, signal sequences from ampl ligand, and signal sequences from secreted polypeptides of the sameor related species, as well as viral secretory leaders, for example, theherpes simplex gD signal.

(ii) Origin of Replication Component

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells.Generally, in cloning vectors this sequence is one that enables thevector to replicate independently of the host chromosomal DNA, andincludes origins of replication or autonomously replicating sequences.Such sequences are well known for a variety of bacteria, yeast, andviruses. The origin of replication from the plasmid pBR322 is suitablefor most Grain-negative bacteria, the 2μ plasmid origin is suitable foryeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV)are useful for cloning vectors in mammalian cells. Generally, the originof replication component is not needed for mammalian expression vectors(the SV40 origin may typically be used only because it contains theearly promoter).

Most expression vectors are “shuttle” vectors, i.e., they are capable ofreplication in at least one class of organisms but can be transfectedinto another organism for expression. For example, a vector is cloned inE. coli and then the same vector is transfected into yeast or mammaliancells for expression even though it is not capable of replicatingindependently of the host cell chromosome.

DNA may also be amplified by insertion into the host genome. This isreadily accomplished using Bacillus species as hosts, for example, byincluding in the vector a DNA sequence that is complementary to asequence found in Bacillus genomic DNA. Transfection of Bacillus withthis vector results in homologous recombination with the genome andinsertion of antibody DNA. However, the recovery of genomic DNA encodingantibody is more complex than that of an exogenously replicated vectorbecause restriction enzyme digestion is required to excise the antibodyDNA.

(iii) Selection Gene Component

Expression and cloning vectors should contain a selection gene, alsotermed a selectable marker. This gene encodes a protein necessary forthe survival or growth of transformed host cells grown in a selectiveculture medium. Host cells not transformed with the vector containingthe selection gene will not survive in the culture medium. Typicalselection genes encode proteins that (a) confer resistance toantibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate,or tetracycline, (b) complement auxotrophic deficiencies, or (c) supplycritical nutrients not available from complex media, e.g., the geneencoding D-alanine racemase for Bacilli.

One example of a selection scheme utilizes a drug to arrest growth of ahost cell. Those cells that are successfully transformed with aheterologous gene express a protein conferring drug resistance and thussurvive the selection regimen. Examples of such dominant selection usethe drugs neomycin (Southern et al., J. Molec. Appl. Genet., 1:327(1982)) mycophenolic acid (Mulligan et al., Science, 209:1422 (1980)) orhygromycin Sugden et al., Mol. Cell. Biol., 5:410-413 (1985)). The threeexamples given above employ bacterial genes under eukaryotic control toconvey resistance to the appropriate drug G418 or neomycin (geneticin),xgpt (mycophenolic acid), or hygromycin, respectively.

Examples of other suitable selectable markers for mammalian cells arethose that enable the identification of cells competent to take up theantibody nucleic acid, such as dihydrofolate reductase (DHFR) orthymidine kinase. The mammalian cell transformants are placed underselection pressure that only the transformants are uniquely adapted tosurvive by virtue of having taken up the marker. Selection pressure isimposed by culturing the transformants under conditions in which theconcentration of selection agent in the medium is successively changed,thereby leading to amplification of both the selection gene and the DNAthat encodes antibody. Amplification is the process by which genes ingreater demand for the production of a protein critical for growth arereiterated in tandem within the chromosomes of successive generations ofrecombinant cells. Increased quantities of antibody are synthesized fromthe amplified DNA.

For example, cells transformed with the DHFR selection gene are firstidentified by culturing all of the transformants in a culture mediumthat contains methotrexate (Mtx), a competitive antagonist of DHFR. Anappropriate host cell when wild-type DHFR is employed is the Chinesehamster ovary (CHO) cell line deficient in DHFR activity, prepared andpropagated as described by Urlaub and Chasin, Proc. Natl. Acad. Sci.USA, 77:4216 (1980). The transformed cells are then exposed to increasedlevels of Mtx. This leads to the synthesis of multiple copies of theDHFR gene, and, concomitantly, multiple copies of other DNA comprisingthe expression vectors, such as the DNA encoding antibody. Thisamplification technique can be used with any otherwise suitable host,e.g., ATCC No. CCL61 CHO-K1, notwithstanding the presence of endogenousDHFR if, for example, a mutant DHFR gene that is highly resistant to Mtxis employed (EP 117,060). Alternatively, host cells (particularlywild-type hosts that contain endogenous DHFR) transformed orco-transformed with DNA sequences encoding antibody, wild-type DHFRprotein, and another selectable marker such as aminoglycoside 3phosphotransferase (APH) can be selected by cell growth in mediumcontaining a selection agent for the selectable marker such as anaminoglycosidic antibiotic, e.g., kanamycin, neomycin, or G418. See U.S.Pat. No. 4,965,199.

A suitable selection gene for use in yeast is the trp1 gene present inthe yeast plasmid YRp7 (Stinchcomb et al., Nature, 282:39 (1979);Kingsman et al., Gene, 7:141 (1979); or Tschemper et al., Gene, 10:157(1980)). The trp1 gene provides a selection marker for a mutant strainof yeast lacking the ability to grow in tryptophan, for example, ATCCNo. 44076 or PEP4-1 (Jones, Genetics, 85:12 (1977)). The presence of thetrp1 lesion in the yeast host cell genome then provides an effectiveenvironment for detecting transformation by growth in the absence oftryptophan. Similarly, Leu2-deficient yeast strains (ATCC No. 20,622 or38,626) are complemented by known plasmids bearing the Leu2 gene.

(iv) Promoter Component

Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to the antibodynucleic acid. Promoters are untranslated sequences located upstream (5′)to the start codon of a structural gene (generally within about 100 to1000 bp) that control the transcription and translation of particularnucleic acid sequence, such as the antibody nucleic acid sequence, towhich they are operably linked. Such promoters typically fall into twoclasses, inducible and constitutive. Inducible promoters are promotersthat initiate increased levels of transcription from DNA under theircontrol in response to some change in culture conditions, e.g., thepresence or absence of a nutrient or a change in temperature. At thistime a large number of promoters recognized by a variety of potentialhost cells are well known. These promoters are operably linked toantibody encoding DNA by removing the promoter from the source DNA byrestriction enzyme digestion and inserting the isolated promotersequence into the vector. Both the native antibody promoter sequence andmany heterologous promoters may be used to direct amplification and/orexpression of the antibody DNA. However, heterologous promoters arepreferred, as they generally permit greater transcription and higheryields of expressed antibody as compared to the native promoter.

Promoters suitable for use with prokaryotic hosts include theβ-lactamase and lactose promoter systems (Chang et al., Nature, 275:615(1978); and Goeddel et al., Nature, 281:544 (1979)), alkalinephosphatase, a tryptophan (trp) promoter system (Goeddel, Nucleic AcidsRes., 8:4057 (1980) and EP 36,776) and hybrid promoters such as the tacpromoter (deBoer et al., Proc. Natl. Acad. Sci. USA, 80:21-25 (1983)).However, other known bacterial promoters are suitable. Their nucleotidesequences have been published, thereby enabling a skilled workeroperably to ligate them to DNA encoding antibody (Siebenlist et al.,Cell, 20:269 (1980)) using linkers or adapters to supply any requiredrestriction sites. Promoters for use in bacterial systems also willcontain a Shine-Dalgarno (S.D.) sequence operably linked to the DNAencoding antibody polypeptide.

Promoter sequences are known for eukaryotes. Virtually all eukaryoticgenes have an AT-rich region located approximately 25 to 30 basesupstream from the site where transcription is initiated. Anothersequence found 70 to 80 bases upstream from the start of transcriptionof many genes is a CXCAAT region where X may be any nucleotide. At the 3end of most eukaryotic genes is an AATAAA sequence that may be thesignal for addition of the poly A tail to the 3 end of the codingsequence. All of these sequences are suitably inserted into eukaryoticexpression vectors.

Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase (Hitzeman et al., J.Biol. Chem., 255:2073 (1980)) or other glycolytic enzymes (Hess et al.,J. Adv. Enzyme Reg., 7:149 (1968); and Holland, Biochemistry, 17:4900(1978)), such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin Hitzeman et al., EP 73,657A. Yeast enhancers also are advantageouslyused with yeast promoters.

Antibody transcription from vectors in mammalian host cells may becontrolled, for example, by promoters obtained from the genomes ofviruses such as polyoma virus, fowlpox virus (UK 2,211,504 publishedJul. 5, 1989), adenovirus (such as Adenovirus 2), bovine papillomavirus, avian sarcoma virus, cytomegalovirus, a netrovirus, hepatitis-Bvirus and most preferably Simian Virus 40 (SV40), from heterologousmammalian promoters, e.g., the actin promoter or an immunoglobulinpromoter, from heat-shock promoters, and from the promoter normallyassociated with the antibody sequence, provided such promoters arecompatible with the host cell systems.

The early and late promoters of the SV40 virus are conveniently obtainedas an SV40 restriction fragment that also contains the SV40 viral originof replication. Fiers et al., Nature, 273:113 (1978); Mulligan and Berg,Science, 209:1422-1427 (1980); Pavlakis et al., Proc. Natl. Acad. Sci.USA, 78:7398-7402 (1981). The immediate early promoter of the humancytomegalovirus is conveniently obtained as a HindIII E restrictionfragment. Greenaway et al., Gene, 18:355-360 (1982). A system forexpressing DNA in mammalian hosts using the bovine papilloma virus as avector is disclosed in U.S. Pat. No. 4,419,446. A modification of thissystem is described in U.S. Pat. No. 4,601,978. See also Gray et al.,Nature, 295:503-508 (1982) on expressing cDNA encoding immune interferonin monkey cells; Reyes et al., Nature, 297:5984-601 (1982) on expressionof human β-interferon cDNA in mouse cells under the control of athymidine kinase promoter from herpes simplex virus; Canaani and Berg,Proc. Natl. Acad. Sci. USA, 79:5166-5170 (1982) on expression of thehuman interferon β1 gene in cultured mouse and rabbit cells; and Gormanet al., Proc. Natl. Acad. Sci. USA, 79:6777-6781 (1982) on expression ofbacterial CAT sequences in CV-1 monkey kidney cells, chicken embryofibroblasts, Chinese hamster ovary cells, HeLa cells, and mouse NIH-3T3cells using the Rous sarcoma virus long terminal repeat as a promoter.

(v) Enhancer Element Component

Transcription of a DNA encoding the antibody of this invention by highereukaryotes is often increased by inserting an enhancer sequence into thevector. Enhancers are cis-acting elements of DNA, usually about from 10to 300 bp, that act on a promoter to increase its transcription.Enhancers are relatively orientation and position independent, havingbeen found 5 (Laimins et al., Proc. Natl. Acad. Sci. USA, 78:993 (1981))and 3 (Luskyet al., Mol. Cell Bio., 3:1108 (1983)) to the transcriptionunit, within an intron (Banerji et al., Cell, 33:729 (1983)), as well aswithin the coding sequence itself (Osborne et al., Mol. Cell Bio.,4:1293 (1984)). Many enhancer sequences are now known from mammaliangenes (globin, elastase, albumin, a-fetoprotein, and insulin).Typically, however, one will use an enhancer from a eukaryotic cellvirus. Examples include the SV40 enhancer on the late side of thereplication origin (bp 100-270), the cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers. See also Yaniv, Nature, 297:17-18(1982) on enhancing elements for activation of eukaryotic promoters. Theenhancer may be spliced into the vector at a position 5 or 3 to theantibody encoding sequence, but is preferably located at a site 5 fromthe promoter.

(vi) Transcription Termination Component

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5 and, occasionally 3 untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding antibody.

(vii) Construction and Analysis of Vectors

Construction of suitable vectors containing one or more of the abovelisted components employs standard ligation techniques. Isolatedplasmids or DNA fragments are cleaved, tailored, and religated in theform desired to generate the plasmids required.

For analysis to confirm correct sequences in plasmids constructed, theligation mixtures are used to transform E. coli K12 strain 294 (ATCC No.31,446) and successful transformants selected by ampicillin ortetracycline resistance where appropriate. Plasmids from thetransformants are prepared, analyzed by restriction endonucleasedigestion, and/or sequenced by the method of Messing et al., NucleicAcids Res., 9:309 (1981) or by the method of Maxam et al., Methods inEnzymology, 65:499 (1980).

(viii) Transient Expression Vectors

Particularly useful in the practice of this invention are expressionvectors that provide for the transient expression in mammalian cells ofDNA encoding the antibody polypeptide. In general, transient expressioninvolves the use of an expression vector that is able to replicateefficiently in a host cell, such that the host cell accumulates manycopies of the expression vector and, in turn, synthesizes high levels ofa desired polypeptide encoded by the expression vector. Sambrook et al.,supra, pp. 16.17-16.22. Transient expression systems, comprising asuitable expression vector and a host cell, allow for the convenientpositive identification of polypeptides encoded by cloned DNAs, as wellas for the rapid screening of such polypeptides for desired biologicalor physiological properties. Thus, transient expression systems areparticularly useful in the invention for purposes of identifyinganalogues and variants of antibody polypeptide that have antibodypolypeptide biological activity.

(ix) Suitable Exemplary Vertebrate Cell Vectors

Other methods, vectors, and host cells suitable for adaptation to thesynthesis of the antibody in recombinant vertebrate cell culture aredescribed in Gething et al., Nature, 293:620-625 (1981); Mantei et al.,Nature, 281:40-46 (1979); Levinson et al.; EP 117,060; and EP 117,058. Aparticularly useful plasmid for mammalian cell culture expression ispRK5 (EP 307,247 U.S. Pat. No. 5,258,287) or pSV16B (PCT Publication No.WO 91/08291).

Suitable host cells for cloning or expressing the vectors herein are theprokaryote, yeast, or higher eukaryotic cells described above. Suitableprokaryotes include eubacteria, such as Gram-negative or Gram-positiveorganisms, for example, E. coli, Bacilli such as B. subtilis,Pseudomonas species such as P. aeruginosa, Salmonella typhimurium, orSerratia marcescans. One preferred E. coli cloning host is E. coli 294(ATCC No. 31,446), although other strains such as E. coli B, E. coliX1776 (ATCC No. 31,537), and E. coli W3110 (ATCC No. 27,325) aresuitable. These examples are illustrative rather than limiting.Preferably the host cell should secrete minimal amounts of proteolyticenzymes. Alternatively, in vitro methods of cloning, e.g., PCR or othernucleic acid polymerase reactions, are suitable.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable hosts for antibody encoding vectors.Saccharomyces cerevisiae, or common baker's yeast, is the most commonlyused among lower eukaryotic host microorganisms. However, a number ofother genera, species, and strains are commonly available and usefulherein, such as Schizosaccharomyces pombe (Beach and Nurse, Nature,290;140 (1981); EP 139,383 published May 2, 1985), Kluyveromyces hosts(U.S. Pat. No. 4,943,529) such as, e.g., K. lactis (Louvencourt et al.,J. Bacteriol., 737 (1983)), K. fragilis, K. bulgarius, K.thermotolerans, and K. marxianus, yarrowia (EP 402,226), Pichia pastoris(EP 183,070; Sreekrishna et al., J. Basic Microbiol., 28:265-278(1988)), Candida, Trichoderma reesia (EP 244,234), Neurospora crassa(Case et al., Proc. Natl. Acad. Sci. USA, 76:5259-5263 (1979)), andfilamentous fungi such as, e.g., Neurospora. Penicillium, Tolypocladium(WO 91/00357 published Jan. 10, 1991), and Aspergilius hosts such as A.nidulans (Ballance et al., Biochem. Biophys. Res. Commun., 112:284-289(1983); Tilburn et al., Gene, 26:205-221 (1983); Yelton ed al., Proc.Natl. Acad. Sci. USA, 81:1470-1474 (1984)) and A. niger (Kelly andHynes, EMBO J., 4:475-479 (1985)).

Suitable host cells for the expression of glycosylated antibody arederived from multicellular organisms. Such host cells are capable ofcomplex processing and glycosylation activities. In principle, anyhigher eukaryotic cell culture is workable, whether from vertebrate orinvertebrate culture. Examples of invertebrate cells include plant andinsect cells. Numerous baculoviral strains and variants andcorresponding permissive insect host cells from hosts such as Spodopterafrugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus(mosquito), Drosophila melanogaster (fruitfly), and Bombyx mori havebeen identified. See, e.g., Luckow et al., Bio/Technology, 6:47-55(1988); Miller et al., Genetic Engineering, Setlow et al., eds., Vol. 8(Plenum Publishing, 1986), pp. 277-279; and Maeda et al., Nature,315:592-594 (1985). A variety of viral strains for transfection arepublicly available, e.g., the L-1 variant of Autographa californica NPVand the Bm-5 strain of Bombyx mori NPV, and such viruses may be used asthe virus herein according to the present invention, particularly fortransfection of Spodoptera frugiperda cells.

Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato,and tobacco can be utilized as hosts. Typically, plant cells aretransfected by incubation with certain strains of the bacteriumAgrobacterium tumefaciens, which has been previously manipulated tocontain the antibody DNA. During incubation of the plant cell culturewith A. tumefaciens, the DNA encoding the antibody is transferred to theplant cell host such that it is transfected, and will, under appropriateconditions, express the antibody DNA. In addition, regulatory and signalsequences compatible with plant cells are available, such as thenopaline synthase promoter and polyadenylation signal sequences.Depicker et al., J. Mol. Appl. Gen., 1:561 (1982). In addition, DNAsegments isolated from the upstream region of the T-DNA 780 gene arecapable of activating or increasing transcription levels ofplant-expressible genes in recombinant DNA-containing plant tissue. EP321,196 published Jun. 21, 1989.

However, interest has been greatest in vertebrate cells, and propagationof vertebrate cells in culture (tissue culture) has become a routineprocedure in recent years (Tissue Culture, Academic Press, Kruse andPatterson, editors (1973)). Examples of useful mammalian host cell linesare monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651);human embryonic kidney line (293 or 293 cells subcloned for growth insuspension culture, Graham et al., J. Gen Virol., 36:59 (1977)); babyhamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovarycells/-DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216(1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251(1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkeykidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells(HELA, ATCC CCL 2); canine kidney cells (MDCIK, ATCC CCL 34); buffalorat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCCCCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci.,383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line(Hep G2).

Host cells are transfected and preferably transformed with theabove-described expression or cloning vectors of this invention andcultured in conventional nutrient media modified as appropriate forinducing promoters, selecting transformants, or amplifying the genesencoding the desired sequences.

Transfection refers to the taking up of an expression vector by a hostcell whether or not any coding sequences are in fact expressed. Numerousmethods of transfection are known to the ordinarily skilled artisan, forexample, CaPO₄ and electroporation. Successful transfection is generallyrecognized when any indication of the operation of this vector occurswithin the host cell.

Transformation means introducing DNA into an organism so that the DNA isreplicable, either as an extrachromosomal element or by chromosomalintegrant. Depending on the host cell used, transformation is done usingstandard techniques appropriate to such cells. The calcium treatmentemploying calcium chloride, as described in section 1.82 of Sambrook etal., supra, is generally used for prokaryotes or other cells thatcontain substantial cell-wall barriers. Infection with Agrobacteriumtumefaciens is used for transformation of certain plant cells, asdescribed by Shaw et al., Gene, 23:315 (1983) and WO 89/05859 publishedJun. 29, 1989. In addition, plants may be transfected using ultrasoundtreatment as described in WO 91/00358 published Jan. 10, 1991. Formammalian cells without such cell walls, the calcium phosphateprecipitation method of Graham and van der Eb, Virology, 52:456-457(1978) is preferred. General aspects of mammalian cell host systemtransformations have been described by Axel in U.S. Pat. No. 4,399,216issued Aug. 16, 1983. Transformations into yeast are typically carriedout according to the method of Van Solingen et al., J. Bact., 130:946(1977) and Hsiao et al., Proc. Natl. Acad. Sci. (USA), 76:3829 (1979).However, other methods for introducing DNA into cells such as by nuclearinjection, electroporation, or protoplast fusion may also be used.

Prokaryotic cells used to produce the antibody polypeptide of thisinvention are cultured in suitable media as described generally inSambrook et al., supra.

The mammalian host cells used to produce the antibody of this inventionmay be cultured in a variety of media. Commercially available media suchas Ham's F10 (Sigma), Minimal Essential Medium ((MEM), Sigma), RPMI-1640(Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) aresuitable for culturing the host cells. In addition, any of the mediadescribed in Ham and Wallace, Meth. Enz., 58:44 (1979), Barnes and Sato,Anal. Biochem., 102:255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866;4,927,762; or 4,560,655; WO 90/03430; WO 87/00195; or U.S. Pat. Re. No.30,985; the disclosures of all of which are incorporated herein byreference, may be used as culture media for the host cells. Any of thesemedia may be supplemented as necessary with hormones and/or other growthfactors (such as insulin, transferrin, or epidermal growth factor),salts (such as sodium chloride, calcium, magnesium, and phosphate),buffers (such as HEPES), nucleosides (such as adenosine and thymidine),antibiotics (such as Gentamycin™ drug), trace elements (defined asinorganic compounds usually present at final concentrations in themicromolar range), and glucose or an equivalent energy source. Any othernecessary supplements may also be included at appropriate concentrationsthat would be known to those skilled in the art. The culture conditions,such as temperature, pH, and the like, are those previously used withthe host cell selected for expression, and will be apparent to theordinarily skilled artisan.

The host cells referred to in this disclosure encompass cells in invitro culture as well as cells that are within a host animal.

Gene amplification and/or expression may be measured in a sampledirectly, for example, by conventional Southern blotting, northernblotting to quantitate the transcription of mRNA (Thomas, Proc. Natl.Acad. Sci. USA, 77:5201-5205 (1980)), dot blotting (DNA analysis), or insitu hybridization, using an appropriately labeled probe, based on thesequences provided herein. Various labels may be employed, most commonlyradioisotopes, particularly ³²P. However, other techniques may also beemployed, such as using biotin-modified nucleotides for introductioninto a polynucleotide. The biotin then serves as the site for binding toavidin or antibodies, which may be labeled with a wide variety oflabels, such as radionuclides, fluorescers, enzymes, or the like.Alternatively, antibodies may be employed that can recognize specificduplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybridduplexes or DNA-protein duplexes. The antibodies in turn may be labeledand the assay may be carried out where the duplex is bound to a surface,so that upon the formation of duplex on the surface, the presence ofantibody bound to the duplex can be detected.

Gene expression, alternatively, may be measured by immunologicalmethods, such as immunohistochemical staining of tissue sections andassay of cell culture or body fluids, to quantitate directly theexpression of gene product. With immunohistochemical stainingtechniques, a cell sample is prepared, typically by dehydration andfixation, followed by reaction with labeled antibodies specific for thegene product coupled, where the labels are usually visually detectable,such as enzymatic labels, fluorescent labels, luminescent labels, andthe like. A particularly sensitive staining technique suitable for usein the present invention is described by Hsu et al., Am. J. Clin. Path.,75:734-738 (1980).

Antibody preferably is recovered from the culture medium as a secretedpolypeptide, although it also may be recovered from host cell lysateswhen directly expressed without a secretory signal.

When the antibody is expressed in a recombinant cell other than one ofhuman origin, the antibody is completely free of proteins orpolypeptides of human origin. However, it is still usually necessary topurify the antibody from other recombinant cell proteins or polypeptidesto obtain preparations that are substantially homogeneous as to the mplligand per se. As a first step, the culture medium or lysate iscentrifuged to remove particulate cell debris. The membrane and solubleprotein fractions are then separated. Alternatively, a commerciallyavailable protein concentration filter (e.g., AMICON or MilliporePELLICON ultrafiltration units) may be used. The antibody may then bepurified from the soluble protein fraction. The antibody thereafter ispurified from contaminant soluble proteins and polypeptides by saltingout and exchange or chromatographic procedures employing various gelmatrices. These matrices include; acrylamide, agarose, dextran,cellulose and others common to protein purification. Exemplarychromatography procedures suitable for protein purification includeimmunoaffinity, receptor affinity (e.g., mpl-IgG or protein ASEPHAROSE), hydrophobic interaction chromatography (HIC) (e.g., ether,butyl, or phenyl Toyopearl), lectin chromatography (e.g., ConA-SEPHAROSE, lentil-lectin-SEPHAROSE), size exclusion (e.g., SEPHADEXG-75), cation- and anion-exchange columns (e.g., DEAE or carboxymethyl-and sulfopropyl-cellulose), and reverse-phase high performance liquidchromatography (RP-HPLC) (see e.g., Urdal et al., J. Chromarog., 296:171(1984) where two sequential RP-HPLC steps are used to purify recombinanthuman IL-2). Other purification steps optionally include; ethanolprecipitation; ammonium sulfate precipitation; chromatofocusing;preparative SDS-PAGE, and the like.

Antibody variants in which residues have been deleted, inserted, orsubstituted are recovered in the same fashion, taking account of anysubstantial changes in properties occasioned by the variation. Forexample, preparation of a an antibody fusion with another protein orpolypeptide, e.g., a bacterial or viral antigen, facilitatespurification; an immunoaffinity column containing antibody to theantigen can be used to adsorb the fusion polypeptide. Immunoaffinitycolumns such as a rabbit polyclonal anti-antibody column can be employedto absorb the antibody variant by binding it to at least one remainingimmune epitope. Alternatively, the antibody may be purified by affinitychromatography using a purified receptor-IgG coupled to a (preferably)immobilized resin such as AFF1-Gel 10 (Bio-Rad, Richmond, Calif.) or thelike, by means well known in the art. A protease inhibitor such asphenyl methyl sulfonyl fluoride (PMSF) also may be useful to inhibitproteolytic degradation during purification, and antibiotics may beincluded to prevent the growth of adventitious contaminants. One skilledin the art will appreciate that purification methods suitable for nativethe antibody may require modification to account for changes in thecharacter of the antibody or its variants upon expression in recombinantcell culture.

In a most preferred embodiment of the invention, the antibodies areagonist antibodies (aAb). By “agonist antibody” is meant an antibodywhich is able to bind to and to activate, a particular hematopoieticgrowth factor receptor. For example, the agonist may bind to theextracellular domain of the receptor and thereby cause differentiationand proliferation of megakaryocyte colonies in semisolid cultures andsingle megakaryocytes in liquid suspension cultures and plateletproduction in vitro and/or in vivo. The agonist antibodies arepreferably against epitopes within the extracellular domain of thereceptor Accordingly, the antibody preferably binds to substantially thesame epitope as the 12E10, 12B5, 10F6, and 12D5 monoclonal antibodiesspecifically disclosed herein. Most preferably, the antibody will alsohave substantially the same or greater antigen binding affinity as themonoclonal antibodies disclosed herein. To determine whether amonoclonal antibody has the same specificity as an antibody specificallydisclosed, one can, for example, use a competitive ELISA binding assay.

DNA encoding the monoclonal antibodies useful in the method of theinvention is readily isolated and sequenced using conventionalprocedures (e.g., by using oligonucleotide probes that are capable ofbinding specifically to genes encoding the heavy and light chains ofhuman antibodies). The phage of the invention serve as a preferredsource of such DNA. Once isolated, the DNA may be placed into expressionvectors, which are then transfected into host cells such as E. colicells, simian COS cells, Chinese Hamster Ovary (CHO) cells, or myelomacells that do not otherwise produce immunoglobulin protein, to obtainthe synthesis of monoclonal antibodies in the recombinant host cells.

IV. Utility

The antibodies disclosed herein are useful for in vitro diagnosticassays for activating the receptor of interest. This is useful in orderto study the role of the receptor in megakaryocyte growth and/ordifferentiation and platelet production.

The biologically active c-mpl agonist antibody capable of stimulatingeither proliferation, differentiation or maturation and/or modulation(either stimulation or inhibition) of apoptosis of hematopoietic cellsmay be used in a sterile pharmaceutical preparation or formulation tostimulate megakaryocytopoietic or thrombopoietic activity in patientssuffering from thrombocytopenia due to impaired production,sequestration, or increased destruction of platelets.Thrombocytopenia-associated bone marrow hypoplasia (e.g., aplasticanemia following chemotherapy or bone marrow transplant) may beeffectively treated with the aAb compounds of this invention as well asdisorders such as disseminated intravascular coagulation (DIC), immunethrombocytopenia (including HIV-induced ITP and non HIV-induced ITP),chronic idiopathic thrombocytopenia, congenital thrombocytopenia,myelodysplasia, and thrombotic thrombocytopenia.

Preferred uses of the megakaryocytopoietic or thrombocytopoieticbiologically active c-mpl agonist antibody of this invention are in:myelotoxic chemotherapy for treatment of leukemia or solid tumors,myeloablative chemotherapy for autologous or allogeneic bone marrowtransplant, myelodysplasia, idiopathic aplastic anemia, congenitalthrombocytopenia, and immune thrombocytopenia.

The biologically active c-mpl agonist antibody of the instant inventionmay be employed alone or in combination with other cytokines,hematopoietins, interleukins, growth factors, or antibodies in thetreatment of the above-identified disorders and conditions. Thus, theinstant compounds may be employed in combination with other protein orpeptide having hematopoietic activity including G-CSF, GM-CSF, LIF,M-CSF, IL-1, IL-3, erythropoietin (EPO), kit ligand, IL-6, and IL-11.

The biologically active c-mpl agonist antibody of the instant inventionmay be used in the same way and for the same indications asthrombopoietin (TPO). Some forms of the aAb have a longer half-life thannative or pegylated TPO and thus are used in indications where a longerhalf-life are indicated.

When used for in vivo administration, the antibody formulation must besterile. This is readily accomplished by filtration through sterilefiltration membranes, prior to or following lyophilization andreconstitution. The antibody ordinarily will be stored in lyophilizedform or in solution.

Therapeutic antibody compositions generally are placed into a containerhaving a sterile access port, for example, an intravenous solution bagor vial having a stopper pierceable by a hypodermic injection needle.

The route of antibody administration is in accord with known methods,e.g., injection or infusion by intravenous, intraperitoneal,intracerebral, intramuscular, intraocular, intraarterial, intrathecal,inhalation or intralesional routes, or by sustained release systems asnoted below. The antibody is preferably administered continuously byinfusion or by bolus injection.

An effective amount of antibody to be employed therapeutically willdepend, for example, upon the therapeutic objectives, the route ofadministration, and the condition of the patient. Accordingly, it willbe necessary for the therapist to titer the dosage and modify the routeof administration as required to obtain the optimal therapeutic effect.Typically, the clinician will administer antibody until a dosage isreached that achieves the desired effect The progress of this therapy iseasily monitored by conventional assays.

The antibodies of the invention may be prepared in a mixture with apharmaceutically acceptable carrier. This therapeutic composition can beadministered intravenously or through the nose or lung, preferably as aliquid or powder aerosol (lyophilized). The composition may also beadministered parenterally or subcutaneously as desired. Whenadministered systematically, the therapeutic composition should besterile, pyrogen-free and in a parenterally acceptable solution havingdue regard for pH, isotonicity, and stability. These conditions areknown to those skilled in the art. Briefly, dosage formulations of thecompounds of the present invention are prepared for storage oradministration by mixing the compound having the desired degree ofpurity with physiologically acceptable carriers, excipients, orstabilizers. Such materials are non-toxic to the recipients at thedosages and concentrations employed, and include buffers such as TRISHCl, phosphate, citrate, acetate and other organic acid salts;antioxidants such as ascorbic acid; low molecular weight (less thanabout ten residues) peptides such as polyarginine, proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidinone; amino acids such as glycine, glutamic acid,aspartic acid, or arginine; monosaccharides, disaccharides, and othercarbohydrates including cellulose or its derivatives, glucose, mannose,or dextrins; chelating agents such as EDTA; sugar alcohols such asmannitol or sorbitol; counterions such as sodium and/or nonionicsurfactants such as TWEEN, PLURONICS or polyethyleneglycol.

Sterile compositions for injection can be formulated according toconventional pharmaceutical practice. For example, dissolution orsuspension of the active compound in a vehicle such as water ornaturally occurring vegetable oil like sesame, peanut, or cottonseed oilor a synthetic fatty vehicle like ethyl oleate or the like may bedesired. Buffers, preservatives, antioxidants and the like can beincorporated according to accepted pharmaceutical practice.

Suitable examples of sustained-release preparations includesemipermeable matrices of solid hydrophobic polymers containing thepolypeptide, which matrices are in the form of shaped articles, e.g.,films, or microcapsules. Examples of sustained-release matrices includepolyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) asdescribed by Langer et al., J. Biomed. Mater. Res., 15:167-277 (1981)and Langer, Chem. Tech., 12:98-105 (1982) or poly(vinylalcohol)),polylactides (U.S. Pat. No. 3,773,919, EP 58,481), copolymers ofL-glutamic acid and gamma ethyl-L-glutamate (Sidman et al., Biopolymers,22:547-556 (1983)), non-degradable ethylene-vinyl acetate (Langer etal., supra), degradable lactic acid-glycolic acid copolymers such as theLUPRON Depot™ (injectable microspheres composed of lactic acid-glycolicacid copolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyricacid (EP 133,988).

While polymers such as ethylene-vinyl acetate and lactic acid-glycolicacid enable release of molecules for over 100 days, certain hydrogelsrelease proteins for shorter time periods. When encapsulated proteinsremain in the body for a long time, they may denature or aggregate as aresult of exposure to moisture at 37° C., resulting in a loss ofbiological activity and possible changes in immunogenicity. Rationalstrategies can be devised for protein stabilization depending on themechanism involved. For example, if the aggregation mechanism isdiscovered to be intermolecular S—S bond formation through disulfideinterchange, stabilization may be achieved by modifying sulfhydrylresidues, lyophilizing from acidic solutions, controlling moisturecontent, using appropriate additives, and developing specific polymermatrix compositions.

Sustained-release compositions also include liposomally entrapped TPO.Liposomes containing TPO are prepared by methods known per se: DE3,218,121; Epstein et al., Proc. Natl. Acad. Sci. USA, 82:3688-3692(1985); Hwang et al., Proc. Natl. Acad. Sci. USA, 77:4030-4034 (1980);EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese patentapplication 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP102,324. Ordinarily the liposomes are of the small (about 200-800Angstroms) unilamellar type in which the lipid content is greater thanabout 30 mol. % cholesterol, the selected proportion being adjusted forthe optimal therapy.

The dosage of the antibody will be determined by the attending physiciantaking into consideration various factors known to modify the action ofdrugs including severity and type of disease, body weight, sex, diet,time and route of administration, other medications and other relevantclinical factors. Therapeutically effective dosages may be determined byeither in vitro or in vivo methods.

An effective amount of the agonist antibody to be employedtherapeutically will depend, for example, upon the therapeuticobjectives, the route of administration, and the condition of thepatient. Accordingly, it will be necessary for the therapist to titerthe dosage and modify the route of administration as required to obtainthe optimal therapeutic effect. A typical daily dosage might range fromabout 1 μg/kg to up to 1000 mg/kg or more, depending on the factorsmentioned above. Typically, the clinician will administer the moleculeuntil a dosage is reached that achieves the desired effect. The progressof this therapy is easily monitored by conventional assays

Depending on the type and severity of the disease, from about 0.001mg/kg to about 1000 mg/kg, more preferably about 0.01 mg to 100 mg/kg,more preferably about 0.010 to 20 mg/kg of the agonist antibody might bean initial candidate dosage for administration to the patient, whether,for example, by one or more separate administrations, or by continuousinfusion. For repeated administrations over several days or longer,depending on the condition, the treatment is repeated until a desiredsuppression of disease symptoms occurs or the desired improvement in thepatient's condition is achieved. However, other dosage regimens may alsobe useful.

EXAMPLES

Without further description, it is believed that one of ordinary skillin the art can, using the preceding description and illustrativeexamples, make and utilize the present invention to the fullest extent.The following working examples therefore specifically point outpreferred embodiments of the present invention, and are not to beconstrued as limiting in any way of the remainder of the disclosure.

Example 1 Assays

The mpl agonist antibody assays were conducted essentially as describedin WO 95/18858.

(a) Ba/F3 Cell Proliferation Assay

The Ba/F3-mpl cell line was established (F. de Sauvage et al., Nature,369:533 (1994)) by introduction of the cDNA encoding the entire mplreceptor into the IL-3 dependent murine lymphoblastoid cell line Ba/F3.Stimulation of proliferation of Ba/F3-mpl cells in response to variousconcentrations of antibodies or TPO was measured by the amount ofincorporation of ³H-thymidine as previously described (F. de Sauvage etal., supra).

(b) CMK Assay for Induction of Platelet Antigen GPII_(b)III_(a)Expression

CMK cells are maintained in RMPI 1640 medium (Sigma) supplemented with10% fetal bovine serum and 10 mM glutamtine. In preparation for theassay, the cells are harvested, washed and resuspended at 5×10⁵ cells/mlin serum-free GIF medium supplemented with 5 mg/l bovine insulin, 10mg/l apo-transferrin, 1×trace elements. In a 96-well flat-bottom plate,the TPO standard or experimental agonist antibody samples are added toeach well at appropriate dilutions in 100 ml volumes. 100 ml of the CMKcell suspension is added to each well and the plates are incubated at37° C., in a 5% CO₂ incubator for 48 hours. After incubation, the plateare spun at 1000 rpm at 4° C. for five minutes. Supernatants arediscarded and 100 ml of the FITC-conjugated GPII_(b)III_(a) monoclonal2D2 antibody is added to each well. Following incubation at 4° C. for 1hour, plates are spun again at 1000 rpm for five minutes. Thesupernatants containing unbound antibody are discarded and 200 ml of0.1% BSA-PBS wash is added to each well. The 0.1% BSA-PBS wash step isrepeated three times. Cells are then analyzed on a FASCAN using standardone parameter analysis measuring relative fluorescence intensity.

(c) KIRA ELISA for Measuring Phospyhorylation of the mpl-Rse.gD ChimericReceptor

The human mpl receptor has been disclosed by Vigon et al., PNAS, USA89:5640-5644 (1992). A chimeric receptor comprising the extracellulardomain (ECD) of the mpl receptor and the transmembrane and intracellulardomain (ICD) of Rse (Mark et al., J. of Biol. Chem. 269(14):10720-10728(1994)) with a carboxyl-terminal flag polypeptide (ie. Rse.gD) was madefor use in the KIRA ELISA described herein.

(i) Capture Agent Preparation

Monoclonal anti-gD (clone 5B6) was produced against a peptide fromHerpes simplex virus glycoprotein D (Paborsky et al., ProteinEngineering 3(6)547-553 (1990)). The purified stock preparation wasadjusted to 3.0 mg/ml in phosphate buffered saline (PBS), pH 7.4 and 1.0ml aliquots were stored at −20° C.

(ii) Anti-phosphotyrosine Antibody Preparation

Monoclonal antiphosphotyrosine, clone 4G10, was purchased from UBI (LakePlacid, N.Y.) and biotinylated using long-armbiotin-N-hydroxysuccinamide (Biotin-X-NHS, Research Organics, Cleveland,Ohio).

(iii) Ligand

The mpl ligand was prepared by the recombinant techniques describedherein. The purified mpl ligand was stored at 4° C. as a stock solution.

KIRA ELISA results for agonist antibodies of the invention are shown inFIG. 9. This assay indicates that the antibodies of the inventionactivate the mpl receptor to a degree similar to the cognate ligand TPO.

(d) TPO Receptor-binding Inhibition Assay

NUNC 96-well immunoplates were coated with 50 μl of rabbit anti-humanIgG Fe (Jackson Labs) at 2 μg/ml in carbonate buffer (pH9.6) overnightat 4° C. After blocking with ELISA buffer (PBS, 1% BSA, 0.2% TWEEN 20),the plates were incubated for 2 hr with conditioned media frommpl-Ig-transfected 293 cells. Plates were washed, and 2.5 ng/mlbiotinylated TPO was added in the presence or absence of variousconcentrations of antibodies. After incubation for 1 hr and washing, theamount of TPO bound was detected by incubation with strepAvidin-HRP(Sigma) followed by TMB peroxidase substrate (Kirkegaard & Perry). Alldilutions were performed in ELISA buffer, and all incubations were atroom temperature. Color development was quenched with H₃PO₄ andabsorbance was read at 450-650 nm.

(e) HU-03 Cell Proliferation Assay

The HU-01 cell line (D. Morgan, Hahnemann University) is derived from apatient with acute megakaryoblastic leukemia and is dependent ongranulocyte-macrophage colony stimulating factor (GM-CSF) for growth.The HU-03 cell line used here was derived from HU-01 cells by adaptationto growth in rhTPO rather than GM-CSF.

HU-03 cells were maintained in RPMI 1640 supplemented with 2%heat-inactivated human male serum and 5 ng/ml rhTPO. Before assay, cellswere starved by removing TPO, decreasing serum concentration to 1%, andadjusting the concentration of cells to 2.5×10⁵ cells/ml, followed byincubation for 16 hr. Cells were then washed and seeded into 96-wellplates at a density of 5×10⁴ cells per well in medium containing TPO orantibodies at various concentrations. Quadruplicate assays wereperformed. 1 μCi 3H-thymidine was added to each well before incubationfor 24 hr. Cells were collected with a Packard cell harvester andincorporation of ³H-thymidine was measured with a Top Count Counter(Packard).

(f) Liquid Suspension Megakaryocytopoeisis Assay

The effect of Mpl agonist antibodies on human megakaryocytopoiesis wasdetermined using a modification of the liquid suspension assaypreviously described (Grant et al, Blood 69:1334-1339 (1997)). Buffycoats were collected from human umbilical cord blood and cells washed inphosphate-buffered saline (PBS) by centrifugation at 120 g for 15 min atroom temperature to remove platelet-rich plasma. Cell pellets wereresuspended in Iscove's modified Dulbecco's medium (IMDM, GIBCO)(supplemented with 100 units per ml penicillin and streptomycin),layered onto 60% percoll (density=1.077 gm/ml, Pharmacia), andcentrifuged at 800 g for 20 min at room temperature. The light-densitymononuclear cells were collected from the interface and washed twicewith IMDM. Cells were seeded at 1×10⁶ cells per ml in IMDM supplementedwith 30% fetal bovine serum (FBS), 100 units per ml penicillin andstreptomycin, and 20 μM 2-mercaptoethanol, into 24-well tissue cultureplates (COSTAR). Serial dilutions of thrombopoletin (TPO) or the Fab′2forms of antibody 12B5 or antibody 12D5 were added to quadruplicatewells; control wells contained no additional supplements. Final volumeswere 1 ml per well. The cultures were grown in a humidified incubator at37° C. in 5% CO₂ for 14 days. Megakaryocytopoiesis was quantified usingradiolabelled murine monoclonal antibody HP1-1D (provided by W. L.Nichols, Mayo Clinic) which has been shown to be specific for the humanmegakaryocyte glycoprotein IIb/IIIa (Grant et al., supra). Cells wereharvested from the tissue culture plates, washed twice with assay buffer(20% FBS, 0.002% EDTA in PBS), and resuspended in 100 μl assay buffercontaining 20 ng iodinated HP1-1D (approximatedly 100,000 cpm). Afterincubation at room temperature for 1 hr, the cells were washed twicewith assay buffer and the cell pellets counted with a gamma counter.

FBS used in this assay was treated with Dextran T40 at 1 mg/ml andcharcoal at 10 mg/ml for 30 min, centrifuged, decanted, filtersterilized and heat inactivated at 56° C. for 30 min.

(g) TPO-Antibody Competitive Binding Assays for HU-03 Cells and HumanPlatelets

HU-03 cells were cultured as described above. Platelet rich plasma (PRP)was prepared by centrifugation of citrated whole blood at 400 g's for 5minutes. Binding studies were conducted within three hours ofcollection. ¹²⁵I-TPO was prepared by indirect iodination (Fielder, P.J., Hass, P., Nagel, M., Stefanich, E., Widmer, R., Bennett, G. L.,Keller, G., de Savage, F. J., and Eaton, D. 1997. Human platelets as amodel for the binding and degradation of thrombopoietin. Blood 89:2782-2788) and yielded a specific activity of 15-50 μCi/μg protein.

In a volume of 110 microliters containing 100 pM iodinated TPO, 2×10⁶washed HU-03 cells in Hank's Balanced Salt Solution, 5 mg/ml bovineserum albumin (HBSSB), or 4×10⁷ platelets in plasma, were incubated at37° C. for 30 minutes with varying concentrations of antibody intriplicate. HU-03 cells were agitated during the incubation period tokeep them in suspension. The reaction mixture was overlayed on 1 ml 20%sucrose-HBSSB and microcentrifuged at 13,500 rpm for five minutes. Thesupernatants were aspirated, tube bottoms containing the cell pelletswere cut off, and cell- or platelet-associated radioactivity wasmeasured with an Iso Data Model 120 gamma counter.

Results for several agonist antibodies on the invention in this assayare shown in FIGS. 10A-F. Longer bars in the graphs indicate greateramounts of bound radiolabeled TPO and less competition by the agonistantibody at a particular concentration.

(h) Affinity Determinations.

The receptor-binding affinities of several Fab fragments were calculated(Lofas & Johnsson, 1990) from association and dissociation rateconstants measured using a BIACORE surface plasmon resonance system(Pharmacia Biosensor). A biosensor chip was activated for covalentcoupling of gD-mpl receptor usingN-ethyl-N′″-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) according to the supplier's (PharmaciaBiosensor) instructions. gD-mpl was buffer-exchanged into 10 mM sodiumacetate buffer (pH 4.5) and diluted to approximately 30 μg/mL. Analiquot (35 μL) was injected at a flow rate of 1 μL/min to achieveapproximately 6400 response units (RU) of coupled protein. Finally, 1Methanolamine was injected as a blocking agent. For kineticsmeasurements, 1.5 serial dilutions of Fab were injected in PBS/Tweenbuffer (0.05% Tween-20 in phosphate buffered saline) at 25° C. using aflow rate of 20 μL/min. Equilibrium dissociation constants, K_(d)'s,from SPR measurements were calculated as k_(off)/k_(on). Standarddeviations, s_(on) for k_(on) and s_(off) for k_(off), were obtainedfrom measurements with >4 protein concentrations (k_(on)) or with >7protein concentrations (k_(off)). Dissociation data were fit to a simpleAB→A+B model to obtain koff +/−s.d. (standard deviation ofmeasurements). Pseudo-first order rate constant (ks) were calculated foreach association curve, and plotted as a function of proteinconcentration to obtain kon +/−s.e. (standard error of fit). Theresulting errors e[K] in calculated K_(d)'s were estimated according tothe following formula for propagation of errors:e[K]=[(k_(on))⁻²(s_(off))²)+(k_(off))²(k_(on))⁻⁴(s_(on))²]^(1/2) wheres_(off) and s_(on) are the standard errors in k_(on) and k_(off),respectively.

Example 2 Isolation of Antibodies From the CAT Library

For construction of a library of antibodies displayed of a phage see thefollowing references: WO 92/01047, WO 92/20791, WO 93/06213, WO93/11236, WO 93/19172, WO 95/01438 and WO 95/15388. Briefly, FIGS. 2 and3 presents a cartoon of the construction of a phage library containing6×10⁹ different clones containing single-chain Fv (scFv) antibodiesfused to gene 3 of a phage. Binding selection against an antigen, inthis case c-mpl, can be carried out as shown in FIG. 4 and described ingreater detail below.

(a) The Antigen

Human c-mpl was cloned as described by F. de Sauvage et al., Nature369:533 (1994).

(b) Phage Selection on Immunotubes

NUNC immunotubes were coated with 2 ml of a solution of 10 microg/ml ofgD-c-mpl in PBS at 4° C. overnight. After rinsing with PBS, tubes wereblocked with 3% dry milk in PBS (MPBS) for 2 hr at room temperature. Forthe first round, 10 μl of C.A.T. antibody phage library containing˜1×10¹² c.f.u. were added to 1 ml MPBS for blocking for 1 hr at roomtemperature. Blocked phage were added to coated tubes, and binding ofphage to antigen allowed to continue for 2 hr at 37° C. on a rotatingwheel. Tubes were washed 6 times with PBS-TWEEN and 6 times with PBS,and phage were then eluted with 100 mM TEA for 10 min at roomtemperature, neutralized with 500 μl of 1 M TRIS (pH 7.4), and stored onice until needed. For subsequent rounds, washing was increased to 20times with PBS-TWEEN, and 20 times with PBS.

Eluted phage were used to infect 5 ml of log phase E. coli TG1 cells andplated on 2YT agar supplemented with 2% glucose and 100 μg/mlcarbenicillin. After overnight growth at 30° C., colonies were scrapedinto 10 ml 2YT. 50 μl of this solution was used to inoculate 25 ml of2YT with carbenicillin and glucose and incubated, shaking, for two hoursat 37° C. Helper phage M13K07 (Pharmacia) were added at an m.o.i. of 10.After adsorption, the cells were pelleted and resuspended in 25 ml of2YT with carbenicillin (100 μg/ml) and kanamycin (50 μg/ml) and growthcontinued at 30° C. for 4 hr. E. coli were removed from the phage bycentrifugation, and 1 ml of these phage (approx. 10¹² c.f.u.) were usedin subsequent rounds of selection.

(c) Antibody Phase Selection Using Streptavidin-coated ParamagneticBeads

The library was also selected using soluble biotinylated antigen andstreptavidin-coated paramagnetic beads (see FIG. 5). gD-c-mpl wasbiotinylated using IMMUNOPURE NHS-biotin (biotiny-N-hydroxy-succinimide,Pierce) according to manufacturer's recommendations.

For the first round of panning, 10 μl of the phage library were blockedwith 1 ml of MPBST (3% dry milk powder, 1×PBS, 0.2% TWEEN 20) for 1 houron a rotating wheel at room temperature. Biotinylated gD-c-mpl was thenadded to a final concentration of 100 nM, and phage were allow to bindantigen for 1 hour at 37° C. on a rotator. Meanwhile, 300 μl ofDYNABEADS M-280, coated with streptavidin (DYNAL) were washed 3 timeswith 1 ml MPBST (using a DYNAL Magnetic Particle Concentrator) and thenblocked for 2 hr at 37° C. with 1 ml fresh MPBST on a rotator. The beadsand were collected with the MPC, resuspended in 50 μl of MPBST, andadded to the phage-plus-antigen solution. Mixing continued on a wheel atroom temperature for 15 min. The DYNABEADS and attached phage were thenwashed a total of 7 times: 3 times with 1 ml is PBS-TWEEN, once withMPBS, followed by 3 times with PBS. Phage were eluted from the beads byincubating 5 min at room temperature with 300 μl of 100 mMtriethylamine. The phage-containing supernatant was removed andneutralized with 150 μl of 1M TRIS-HCl (pH 7.4). Neutralized phage wereused to infect mid-log TG1 host cells as described above. Plating,induction and harvesting of phage were also as for selection on tubes.

For the second and subsequent rounds of selection on biotinylatedgD-c-mpl, 1 ml of harvested phage (approximately 10¹² cfu) were blockedwith 200 μl 10% dry milk, 6×PBS, 0.3% TWEEN 20. Antigen concentrationwas decreased at each round of selection. In one series theconcentrations were: first round, 100 nM; second round, 10 nM; thirdround, 1 nM. A second panning was performed using: first round 100 nM;second round 100 nM; third round, 50 nM; fourth round, 10 nM; and fifthround, 1 nM. Washing stringency was increased to two cycles of 7 washesfor rounds 2, and three cycles for rounds 3 and beyond.

(d) ELISA Screening of Selected Clones

After each round of selection, individual carbenicillin-resistantcolonies were screened by ELISA to identify those producingc-mpl-binding phage. Only those clones which were positive in two ormore assay formats were further studied. FIG. 6 illustrates the phageELISA process.

Individual clones were inoculated into 2TY with 2% glucose and 100 μg/mlcarbenicillin in 96-well tissue culture plates and grown until turbid.Cultures were then infected at an m.o.i. of 10 with M12KO7 helper phage,and infected cells were transferred to 2YT media containingcarbenicillin (100 μg/ml) and kanamycin (50 μg/ml) for growth overnightat 30° C. with gentle shaking.

NUNC MAXISORP microtiter plates were coated with 50 μl per well ofgD-c-mpl, BSA, or gD-gp120, at 2 μg/ml in 50 mM carbonate buffer (pH9.6), at 4° C. overnight. After removing antigen, plates were blockedwith 3% dry milk in PBS (MPBS) for 2 hours at room temperature.

Phage cultures were centrifuged and 100 μl of phage-containingsupernatants were blocked with 20 μl of 6×PBS/18% dry milk for 1 hour atroom temperature. Block was removed from titer plates and blocked phageadded and allowed to bind for 1 hour at room temperature. After washing,phage were detected with a 1:5000 dilution of horseradishperoxidase-conjugated anti-M13 antibody (Pharmacia) in MPBS followed by3′,3′,5′,5′-tetramethylbenzidine (TMB). Reactions were stopped by theaddition of H₂SO₄ and readings taken by subtracting the A_(405nm) fromthe A_(450nm).

(e) Soluble scFv ELISA

Soluble scFv was induced in the bacterial supernatants of clones bygrowth in 2YT containing carbenicillin (100 μg/ml) and IPTG (1 mM) ON at30° C. ELISA plates were either coated with gD-c-mpl or, for captureELISA, with anti-c-myc Mab 9E10. Plates were blocked with 1×ELISAdiluent (PBS supplemented with 0.5% BSA, 0.05% Tween 20, pH 7.4), andsoluble scFv was blocked by adding 20 μl of 6×ELISA dil to 100 μl ofsupernatant. After binding to antigen coated plates, soluble scFv wasdetected by adding 50 μl of 1 μg/ml Mab 9E10 per well, followed byhorseradish peroxidase-conjugated goat anti-murine Ig, and then TMB asdescribed above. For capture ELISA, soluble scFv was detected byaddition of biotinylated c-mpl, followed by streptavidin-peroxidaseconjugate and then TMB as above.

The number of clones screened by ELISA from each round, and the numberof clones positive by phage ELISA are shown in Table 3.

TABLE 3 Anti-c-mpl scFv antibodies from CAT library Clones screened:1534 Clones positive by ELISA: 361 Clones different by BstNl andsequencing: 24 Clones that express protein well 17 clones that areagonists by KIRA: 9 clones that are agonists by BaF3 proliferationassay: 6 clones that are agonists by Hu3 proliferation assay: 4

(f) DNA fingerprinting of clones

The diversity of c-mpl-binding clones was determined by PCR amplifyingthe scFv insert using primers pUC19R (5 AGC GGA TAA CAA TTT CAC ACA GG3) (SEQ. ID. NO: 54) which anneals upstream of the leader sequence andfdtetseq (5 GTC GTC TTT CCA GAC GGT AGT 3) (SEQ. ID. NO: 55) whichanneals in the 5 end of gene III, followed by digestion with thefrequent-cutting restriction enzyme BstNI (see FIG. 7).

Typical patterns seen after analysis on a 3% agarose gel are shown inFIGS. 8A-C.

DNA Fingerprinting: Protocol

Mix A: dH2O 67 μl 10 x ampliTaq buffer 10 25 mM MgCl2 10 DMSO, 50% 2forward primer 1 Mix B: 2.5 mM dNTPs 8 μl AMPLITAQ 0.5 reverse primer1.0

Place 90 μl of Mix A in reaction tube

Inoculate with very small portion of E.coli colony using a yellow tip

Heat in PCR block to 98° C., 3 min. Remove to ice.

Add 10 μl Mix B

Cycle: 95° C., 30 sec, 55° C. 30 see, 72° C. 1 min 20 sec, for 25cycles, in Perkin Elmer 2400

Remove 10 μl ro run on a 1% agarose gel, test for a 1 kB band

Make remaining mix to 1×BstNI reaction buffer

Add 5 units BstNI

60° C., 2 hours

Electrophorese samples on 3% METAPHORE agarose gel

(g) Sequencing of Clones

The nucleotide sequence of representative clones of each fingerprintwere obtained. Colonies were inoculated into 50 ml of LB mediumsupplemented with 2% glucose and 100 μg/ml carbenicillin, and grownovernight at 30° C. DNA was isolated using Qiagen Tip-100s and themanufacturer's protocol and cycle sequenced with fluorescent dideoxychain terminators (Applied Biosystems). Samples were run on an AppliedBiosystems 373A Automated DNA Sequencer and sequences analyzed using theprogram “Sequencher” (Gene Codes Corporation). The VH and VL genes wereassigned to a germline segment using the antibody database, V-BASE.

DNA sequence was obtained for 39 clones and resulted in 24 differentc-mpl-binding scFvs.

(h) Purification of scFvs With (his)₆

For protein purification of soluble antibody, E. coli strain 33D3 wastransformed with phagemid DNA. Five ml of 2YT with carbenicillin andglucose was used to grow overnight cultures at 30° C. 0.2 ml of thesecultures were diluted into 200 ml of the same media and grown to anOD₆₀₀ of approximately 0.9. The cells were pelleted and resuspended in250 ml of 2YT containing IPTG (1 mM) and carbenicillin (100 μg/ml) andto induce expression and grown for a further 5 hours at 30° C. Cellpellets were harvested and frozen at −20° C.

The antibodies were purified by immobilized metal chelate affinitychromatography (IMAC). Frozen pellets were resuspended in 10 ml ofice-cold shockate buffer (25 mM TRIS-HCl, 1 mM EDTA, 200 mM NaCl, 20%sucrose, 1 mM PMSF) by shaking on ice for 1 hr. Imidazole was added to20 mM, and cell debris removed by centrifugation. The supernatants wereadjusted to 1 mM MgCl₂ and 50 mM phosphate buffer pH 7.5. Ni-NTA agaroseresin from Qiagen was used according to the manufacturers instructions.The resin was equilibrated with 50 mM sodium phosphate buffer pH 7.5,500 mM NaCl, 20 mM imidazole, and the shockate added. Binding occurredin either a batch mode or on a gravity flow column. The resin was thenwashed twice with 10 bed volumes of equilibration buffer, and twice withbuffer containing imidazole increased to 50 mM. Elution of proteins waswith 50 mM phosphate buffer pH 7.5, 500 mM NaCl and 250 mM imidazole.Excess salt and imidazole was removed on a PD-10 column (Pharmacia), andproteins were concentrated using a Centricom 10 to a volume of about 1ml.

Concentration was estimated spectrophotometrically assuming an A280 nmof 1.0=0.6 mg/ml.

Portions of these protein preparations were submitted for KIRA assay,c-mpl-Ba/F3 cell proliferation assay, and Hu3 cell proliferation assay.

Plasmid DNA for scFv clones 12B5, 12D5, 12E10, 10D10, 10F6 and 5E5(named pMpl.12B5.scFv.his; pMpl.12D5.scFv.his; pMpl.12E10.scFv.his;pMpl.10D10.scFv.his; pMpl.10F6.scFv.his; and pMpl.5E5.scFv.his,respectively) has been deposited with ATCC, Manassas, Va., USA.

(i) Reformatting of Antibodies to scFv With gD tag, Fab′, Fab′2, andFull Length Molecules.

For improved expression of scFv, and for Fab′, and Fab′2 forms ofantibodies, some of the anti-c-mpl clones were cloned into derivativesof the expression vector pAK19 (Carter et al. METHODS: A companion toMethods in Enzymology. 3:183-192 (1991). Expression is under thetranscriptional control of the E. coli alkaline phosphatase (phoA)promoter (Chang, et al Gene 44:121-125 (1986) which is inducible byphosphate starvation. Each peptide chain is preceded by the E. colienterotoxin II (stII) signal sequence (Picken, et al.) to directsecretion to the periplasmic space of E. coli. This vector also containsthe human k₁ C_(L) (Palm et al., Infect. Immun. 42:269-275 (1983)) andthe human IgG1 C_(H)1 (Ellison, et al, Nucleic Acids Res. 10:4071-4079(1982)) constant domains. The C_(H)1 gene is immediately followed by thebacteriophage λ t₀ transcriptional terminator (Scholtissek and GrosseNucleic Acids Res. 15: 3185 (1987)).

(i) Fab′ and Fab′2 Construction

Construction of the Fab′ and Fab′2 variants was facilitated by insertioninto pAK19 of unique restriction sites at the junctions of the stII andV_(L) domain (Sfi I), the V₁ and Ck domains (Rsr II), the stII and V_(H)domain (MluI), and the V_(H) and C_(H)1 domains (Apa I), usingoligonucleotide directed mutagenesis. In order to insure expression ofmonovalent Fab′ molecules, the free cysteine at the 3′ end of the CH1domain was mutated to a threonine, these Fab′ molecules thus end in theamino acid sequence thr-ala-ala-pro, rather than thr-cys-ala-ala as inpAK19. This vector for the expression of Fab′ molecules is namedpXCA730.

Since some of the antibodies derived from the library had light chainswhich were derived from lambda rather than kappa light chain families,the human λ C_(L) was subcloned from pB11.2 (Carter, P, Garrard, L.,Henner, D. 1991. Methods: A Companion to Methods in Enzymology.3:183-192) into a derivative of pXCA730 to give vector pXCA970.

For expression of the antibodies as Fab′2 molecules, a vector wasconstructed which adds the human IgG1 hinge region onto the C_(H)1domain of pXCA730. This is followed by the yeast GCN4 leucine zipperdomain (Hu, et al. Science 250:1400-1403 (1990)) for stability. TheseDNA fragments were constructed using synthesized oligonucleotides andencode the amino acid sequence:cys-pro-pro-cys-ala-pro-glu-leu-leu-gly-gly-arg-met-lys-gln-leu-glu-asp-lys-val-glu-glu-leu-leu-ser-lys-asn-tyr-his-leu-glu-asn-glu-val-ala-arg-leu-lys-lys-leu-val-gly-glu-arg(SEQ. ID. NO: 56). The resultant plasmid is named pXCA740.

The variable domains of the scFvs were amplified and restriction sitesadded for subcloning into the vectors described above by the PCRtechnique. Specific oligonucleotides were designed for each V_(L) orV_(H) region as shown below.

12B5, 12D5, and 10D10 Light chain variable domains:

5 primer GCT TCT GCG GCC ACA CAG GCC TAC GCT GAC ATC GTG ATG ACC C (SEQ.ID. NO: 57)

3 primer ATG ATG ATG TGC CAC GGT CCG TTT GAT CTC CAG TTC GGT C (SEQ. ID.NO: 58)

12E10 Light chain variable domain:

5 primer GCT TCT GCG GCC ACA CAG GCC TAC GCT TCC TAT GTG CTG ACT C (SEQ.ID. NO: 59)

3 primer CCT TCT CTC TTT AGG TTG GCC AAG GAC GGT CAG CTT GGT C (SEQ. ID.NO: 60)

10F6 Light chain variable domain

5 primer GCT TCT GCG GCC ACA CAG GCC TAC GCT CAG TCT GTG CTG ACT C (SEQ.ID. NO: 61)

3 primer CCT TCT CTC TTT AGG TTG GCC AAG GAC GGT CAG CTT GGT C (SEQ. ID.NO: 60)

12B5 Heavy chain variable domain

5 primer CAT TCT ACA AAC GCG TAC GCT CAG GTG CAG CTG GTG CAG (SEQ. ID.NO: 62)

3 primer GTA AAT GTA TGG GCC CTT GGT GGA GGA GGC ACT CGA GAC GGT GAC(SEQ. ID. NO: 63)

12D5 Heavy chain variable domain

5 primer CAT TCT ACA AAC GCG TAC GCT CAG GTG CAG CTG GTG GAG (SEQ. ID.NO: 64)

3 primer GTA AAT GTA TGG GCC CTT GGT GGA GGA GGC ACT CGA GAC GGT GAC(SEQ. ID. NO: 63)

10D10 Heavy chain variable domain

5 primer CAT TCT ACA AAC GCG TAC GCT GAC GTG CAG CTG GTG CAG (SEQ. ID.NO: 65)

3 primer GTA AAT GTA TGG GCC CTT GGT GGC GGC TGA GGA GAC GGT GAC (SEQ.ID. NO: 66)

12E10 Heavy chain variable domain

5 primer CAT TCT ACA AAC GCG TAC GCT CAG GTG CAG CTG CAG CAG (SEQ. ID.NO: 67)

3 primer GTA AAT GTA TGG GCC CTT GGT GGA GGA GGC ACT CGA GAC GGT GAC(SEQ. ID. NO: 63)

10F6 Heavy chain variable domain

5 primer CAT TCT ACA AAC GCG TAC GCT CAG GTG CAG CTG CAG GAG (SEQ. ID.NO: 68)

3 primer GTA AAT GTA TGG GCC CTT GGT GGA GGC TGA AGA GAC GGT AAC (SEQ.ID. NO. 69)

PCR reactions were carried out using 100 ng of plasmid DNA containingthe scFv, 0.5 μM of the appropriate 5 and 3 primer, 200 μM each dNTP, 10mM KCl, 6 mM(NH₄)₂SO₄, 20 mM TRIS-HCl, pH 8.0, 2 mM MgCl₂, 1% TritonX-100, 100 μM BSA and 2.5 units of Pfu DNA polymerase (Stratagene).Amplification was for 30 cycles of: 30 sec at 95° C., 30 sec at 55° C.,30 sec at 72° C. After digestion with the appropriate restrictionenzymes, the reaction products were separated by agarose gelelectrophoresis and the approximately 350 bp band was isolated using aGene Clean II kit (BIO 101, Vista, Calif.). The fragments for the lightchain variable regions were ligated into the vectors previously digestedwith Sfi I and Rsr II for the kappa isotypes, or Sfi I and Msc I for thelambda isotypes, and transformed into E. coli DH5a. Desired recombinantswere identified using restriction enzyme analysis and sequenced toconfirm the presence of the desired fragments. The heavy chain variabledomains were then cloned similarly into the plasmids containing thelight chains using the restriction enzymes Mlu I and Apa I, and thefinal constructions were again checked by DNA sequencing.

(k) Construction of scFv With gD Tags.

For increased and regulated expression in high density fermentationtanks, the Sfi I to Not I fragments of the scFv forms of p12B5, p12D5,p10F6, and p12E10 were subcloned into a derivative of pAK19 containingthe phoA promoter and stII signal sequence rather than the lacZ promoterand hybrid signal sequence of the original library. For ease ofpurification, a DNA fragment coding for 12 amino acids(met-ala-asp-pro-asn-arg-phe-arg-gly-lys-asp-leu) (SEQ. ID. NO: 70)derived from herpes simplex virus type I glycoprotein D (Lasky andDowbenko DNA (N.Y.) 3:23-29 (1984.)) was synthesized and inserted at the3 end of the V_(L) domain in place of the (his)₆ and c-myc epitopeoriginally present in the C.A.T. library clones.

(l) Expression in E. coli

Plasmids containing genes for scFv-gD, Fab′ or Fab′2 molecules wereexpressed in E. coli strain 33B6 (W3110 DfhuA phoADE15 deoC2ilvG2096(val^(R)) degP41(DPstI-Kan^(R)) D(argF-lac)169 IN(rrnD-rrnE)1)grown for approximately 40 hr at 30° C. in an aerated 10-liter fermentoras described previously (Carter et al Bio/Technology 10:163-167(1992.)).

Example 3 Cloning and Expression of Full Length Human AntibodyDerivatives of 12B5, 12D5, and 12E10

For expression of full length antibodies in mammalian cells, the heavychain variable domains were subcloned from the Fab constructs into aderivative of expression vector pRK (Suva et al., Science 237:893-896(1987)) which contains the human IgG1 CH1, CH2, and CH3 domains and ahuman antibody signal sequence (Carter et al., Proc. Natl. Acad. Sci.USA. 89:4285-4289 (1992)). The light chain was cloned into a separatepRK plasmid. The light and heavy chain expression vectors werecotransfected into adenovirus-transformed human embryonic kidney cellline 293 by a high-efficiency procedure (Gorman et al, DNA Protein Eng.Technol. 2:3-10 (1990)). Harvested conditioned media was shown tocontain anti-mpl antibody by ELISA.

For production of a more stable cell line and high-level antibodyproduction, the light and heavy chains were moved into the SVI.DIexpression vector previously described (Lucas et al., Nucleic Acids Res.24: 1774-1779 (1996). This vector contains the mouse DHFR cDNA in theintron of the expression vector pRK and allows for amplification ofexpression by selection in methotrexate The light chain is cloned intothe same plasmid with expression driven by a second SV40promoter/enhancer. The plasmid was linearized and transfected into CHOcells using lipofectamine (Gibco-BRL) following manufacturer'sinstructions. Seven to ten days after transfer to selective medium,clones were isolated into 96 well plates for later study, or pooled andexpanded for culture in roller bottles.

Conditioned media for purification of the antibodies was generated inroller bottles. Cells were seeded into the roller bottles at an initialcell density of 2×107 cells in 200 ml rich medium (DMEM: Ham's F12 (1:1)supplemented with 5% fetal bovine serum. At approximately 80%confluency, the media was replaced with serum-free PS-24 productionmedium supplemented with insulin (10 μg/ml), transferrin (10 μg/ml),trace elements and lipid alcohol. Conditioned media was harvested after10 days.

Example 4 Purification of Agonist Antibodies

(a) Purification of scFv With FD Tag

Frozen cell paste was resuspended at 1 gm/ml TE (25 mM TRIS, 1 mM EDTA,pH 7.4) and gently agitated 18 hr on ice. Cell debris was removed bycentrifugation at 10,000×g for 30 min. The supernatant was loaded ontoan affinity column (2.5×9.0 cm) consisting of an anti-gD monoclonalantibody 5B6 (Paborsky, L. R. et al., Protein Eng. 3: 547-553 (1990))coupled to CNBr SEPHAROSE which had been equilibrated with PBS. Thecolumn was washed 18 hr with PBS, and then washed with PBS containing 1M NaCl until the absorbance of the column effluent was equivalent tobaseline. All steps were done at 4° C. at a linear flow rate of 25cm/hr. Elution was performed with 0.1 M acetic acid, 0.5 M NaCl, pH 2.9.Column fractions were monitored by absorbance at 280 nm and peakfractions pooled, neutralized with 1.0 M TRIS, pH 8.0, dialyzed againstPBS, and sterile filtered. The resultant protein preparations wereanalyzed by non-reducing SDS-PAGE.

(b) Purification of Fab′ Molecules

For purification of Fab′ molecules, 5 g of frozen cell paste wasresuspended in 5 ml of TE (25 mM TRIS, 1 mM EDTA, pH 7.4) and gentlystirred 18 hr on ice. The pH of the shockate was adjusted to 5.6 with 2M HCl and the precipitate and cell debris removed by centrifugation at10,000×g for 30 min. The supernatant was loaded onto a 1 ml BAKERBONDABx column (0.5×5.0 cm) (J. T. Baker, Phillipsburg, N.J.)pre-equilibrated with 20 mM MES, pH 5.5. After washing with 20 mM MES tobaseline, the Fab′ was eluted using a 10 ml linear gradient from 0 to100% of 20 mM NaOAc, 0.5 M (NH₄)₂SO₄, pH 7.2, with a flow rate of 153cm/hr. Fractions containing Fab′ were pooled, and buffer exchanged intoPBS.

(c) Purification of Fab′2 Molecules

Frozen cell paste (100 gm) was thawed into 10 volumes of 25 mM TRIS, 5mM EDTA, 1 mM NaN3, pH 7.4 and disrupted by three passages through amicrofluidizer (TECH-MAR). PMSF was added to 1 mM and the cell debrisremoved by centrifugation at 10,000×g for 30 min. The supernatant wasfiltered sequentially through a 0.45 μm, and a 0.2 μm SUPORCAP filter(Gelman), and loaded onto a 50 ml SEPHAROSE-fast-flow Protein-G column(Pharmacia) pre-equilibrated with PBS. After washing to baseline withPBS, Fab′2 was eluted with 0.1 M glycine ethyl ester, pH 2.3, into tubeswith contained 1/10 volume of 1 M TRIS, pH 8.0. Fractions containingFab′2 were pooled and concentrated by Ultrasette with a 30 kilodaltonmolecular weight cut off, and buffer exchanged into 20 mM NaOAc, 0.01%octylglucoside, pH 5.5. This material was loaded onto a 30 mlS-SEPHAROSE column (Pharmacia) pre-equilibrated with 20 mM NaOAc, washedto baseline with 20 mM NaOAc, pH 5.5, and eluted with a linear gradientof 0-1 M NaCl in 25 mM NaOAc over 10 column volumes. Fractionscontaining Fab′2 were pooled and buffer exchanged to PBS.

(d) Purification of Full Length Antibodies From Transfected CHO CellSupernatants.

Conditioned medium harvested from roller bottles was loaded onto a 5 mlProtein-A SEPHAROSE column (1.0×5.0 cm) pre-equilibrated with PBS,washed with PBS, and then washed to baseline with PBS containing 1 MNaCl. Antibody was eluted with 0.1 M HOAc, 0.5 N NaCl, pH 2.9,neutralized with 1 M TRIS, and buffer exchanged to PBS.

A summary of agonist antibody activities for several antibodies andfragments thereof is shown in Table 4 below.

TABLE 4 Summary of Mpl Agonist Antibody Activities Hu3 Pro- Hu3 Mpl/IPOMK Anti- liferation KIRA Binding ELISA Platelets As- body (ED50) (ED50)(IC50) (IC50) (IC50) say I2B5 scFv 20 pM 1 nM  10 nM 17 nM 100 nM ++ Fab<1 μM 3 nM 900 nM none <1 μM − Fab′2 5 pM 1 nM  5 nM 1 nM 300 nM + IgG30 pM 400 pM  10 nM 152 pM 300 nM − I2E10 scFv 5 pM 60 pM  5 nM 1.6 nM 5nM Fab <1 μM <1 μM 500 nM 180 nM <1 μM Fab′2 <1 μM 160 pM  10 nM 640 nM500 nM IgG <1 μM 480 pM  50 nM 450 pM 500 nM I2D5 scFv 1.2 nM 280 pM  10nM 24 nM <1 μM Fab <1 μM 4 nM 500 nM 1 μM <1 μM Fab′2 4.8 pM 600 pM  4nM 1 nM 100 nM + IgG <1 μM 3 nM  10 nM 450 pM 500 nM

Example 5

In another embodiment, the invention provides a method of selecting anantibody which binds to and dimerizes a receptor protein. In thismethod, a library of antibodies is panned using a receptor proteinhaving two protein subunits as the binding target. The library is pannedas described above for mpl agonist antibodies. Preferably, theantibodies are human and more preferably monoclonal. The library isconveniently a library of single chain antibodies, preferably displayedon the surface of phage. The display of proteins, including antibodies,on the surface of phage is well known in the art as discussed above andthese known methods may be used in this invention. Antibody librariesare also commercially available, for example, from Cambridge AntibodyTechnologies (CAT), Cambridge, UK. Preferably, the antibody selected bythe method of the invention activates the receptor by dimerizing thereceptor and thereby achieves an effector result similar to the effectorresult generated when the natural endogenous ligand for the receptorbinds the receptor.

The method of the invention can be used to find agonist antibodies toany receptor having two components which is known and for can be cloned.It is not necessary to know the primary, secondary or tertiary structureof the receptor protein, although this information is useful forcloning, etc., since the method of the invention allows selection ofantibodies which will bind any displayed receptor which is activated bydimerization. Many known receptor proteins are activated by dimerizationand any of these known receptors may be used in the invention. Suitablereceptors include tyrosine kinase receptors and hematopoietic receptorsthat lack kinase activity.

Activation of a receptor such as a tyrosine kinase receptor by a scFv isan unexpected result. Current understanding of receptor activationargues that for many classes of receptors, including tyrosine kinasereceptors and hematopoietic cytokine receptors that lack intrinsictyrosine kinase activity (but associate with intracellular kinases), itis a dimerization event mediated by a ligand that is the key event inreceptor activation. This view is supported by crystal structures ofreceptor ligand complexes as well as the demonstrated agonist ability ofcertain monoclonal antibodies (but not the Fab′ fragments of theseantibodies). A single chain antibody would not, therefore, be expectedto be able to cause receptor dimerization and activation.

MuSK is a recently identified tyrosine kinase localized to thepostsynaptic surface of the neuromuscular junction. (Valenzuela et. al.,1995. Neuron 15 573-584.) Mice made deficient in MuSK fail to formneuromuscular junctions (Dechiara et. al., 1996. Cell 85 501-512.), aphenotype highly similar to that observed in mice lacking the nervederived signaling molecule agrin (Gautam et. al., 1996, Cell 85525-535). The likely involvement of MuSK in agrin signaling isstrengthened by the observations that agrin induces the rapid tyrosinephosphorylation of MuSK and that labeled agrin can be chemicallycrosslinked to MuSK (Glass et. al., 1996, Cell 85 513-523.).

Formation of the neuromuscular junction is achieved through a processthat includes the differentiation of membrane on the muscle fiberproximal to the neuron terminus and changes in gene expression withinthe nuclei proximal to this junction (reviewed by Bowe et. al., 1995,Annu-Rev-Neurosci. 18 443-462 and Kleiman et. al., 1996, Cell 85461-464.). A striking feature of this complex process is theredistribution and concentration of AChRs within the myotube membrane.Agrin is able to the induce this clustering of AChRs as well as changesin the extracellular matrix and cytoskeletal components of the synapticapparatus (Bowe et. al., supra; Godfrey et al., 1984, J. Cell Biol. 99615-627; Nitkin et. al., 1987, J. Cell Biol. 105 2471-2478). Agrin is asecreted protein with a core molecular weight of ˜200 kDa that containsseveral copies of EGF repeats, laminin-like globular domains andsequences that resemble protease inhibitors. It is released by motorneuron terminals and maintained within the basil lamina of the synapticcleft. While agrin apparently does not to bind MuSK with high affinity(Glass et. al., supra), it has been reported to interact with othermolecules present at the neuromuscular junction, most notablyalpha-dystroglycan (O'Toole et. al., 1996,. Natl. Acad. Sci. USA 937369-7374) thereby complicating the analysis of MuSK's role in thesignaling events initiated by agrin.

Antigen specific scFv, identified by panning a diverse library of scFvexpressed, for example, on M13 phage provide a source of moleculescapable of mediating specific therapeutic activities, and offer a rapidnew approach to study the function of novel or recently identifiedmolecules such as MuSK. scFv are identified below that mediate receptoractivation and that direct MuSK activation induces changes in AChRdistribution and tyrosine phosphorylation similar to that observed withagrin.

The induction of AChR clustering and tyrosine phosphorylation by scFvantibodies provides direct evidence to support conclusions drawn fromstudies of knockout mice deficient in MuSK indicating this recentlydiscovered tyrosine kinase acts to induce key events in the formation ofthe neuromuscular junction. As a potential signal transducer of agrin,it is noteworthy that MuSK does not display high affinity binding toagrin, leading to speculation that there must be an additional agrinbinding component(s) involved in mediating the agrin signal. Themolecular nature of this component is unknown. It is interesting that itis possible to induce the receptor clustering, the hallmark activity ofagrin, with an agent directed specifically to MuSK.

The marked upregulation of MuSK expression in muscle followingdenervation or muscle immobilization as well as the chromosomallocalization of MuSK within a region associated with fukiyama musculardystrophy point to an important role for this molecule in regulation ofthe neuromuscular junction (Valenzuela et. al., supra) and indicates thepossibility that therapeutic benefit is possible through the controlledregulation of MuSK activity. As agrin is expressed not only at theneuromuscular junction, but in a wide variety of peripheral and centralneurons (Bowe et. al., supra, Rupp et. al., 1991, Neuron 6 811-823; Tsimet. al., 1992, Neuron 8 677-689) it may not be an optimal candidatemolecule through which to manipulate MuSK function as exogenouslyintroduced agrin derivatives might elicit consequences not restricted tothe neuromuscular junction. Thus, in comparison, the ability to obtaindirect activation of MuSK through scFv offers an attractive alternative.Each of the scFv that were tested displayed affinity for MuSK in the nMrange demonstrating the utility of phage displayed scFv libraries as arich source of high affinity and highly specific molecules.

The antibodies of the invention are, therefore, useful in assaying theupregulation of MuSK receptors in sample tissues to determine the degreeof neuromuscular damage associated with this upregulation. Theantibodies are also useful for activating the MuSK receptor and inducingAChR clustering at neuromuscular junctions as a direct result of theagonist properties of these antibodies. Administration of the antibodiesto a person suffering from denervation or muscle immobilization, e.g.muscular dystrophy, provides a method of improving the function of theneuromuscular junctions in these people.

To prepare scFv having agonist activity, antibodies were selected whichinduce a proliferative response in a factor dependent cell line througha chimeric MuSK-Mpl receptor comprised of the extracellular domain ofMuSK and the intracellular domain of the hematopoietic cytokine receptorc-Mpl (the receptor for thrombopoietin, TPO). Activation of c-Mpl isbelieved to require homodimerization, as is the case for the growthhormone receptor, the erythropoietin receptor and other relatedreceptors of this class (Carter et. al., 1996, Annu-Rev-Physiol 58187-207; Gurney et. al., 1995, Proc. Natl. Acad. Sci. USA 92 5292-5296).Ba/F3 cells expressing MuSK-Mpl were starved of IL-3 and exposed to arange of concentrations of each scFv expressed as soluble protein.Surprisingly, 4 of the 21 scFv were able to induce a robustproliferative response in the MuSK-Mpl expressing cells (FIG. 11). Thisactivity was observed at nM concentrations of scFv. The scFv werewithout effect on the parental, untransfected Ba/F3. Agonist activitywas also present among those IgG that were derived from agonist scFv butwas not noted among IgG derived from non-agonist scFv. Soluble agrinc-terminal domain (c-agrin) was without effect supporting previousobservations that agrin does not bind MuSK directly. The c-terminaldomain of agrin is known to contain the AChR clustering activity ofagrin and is essential for neuromuscular junction formation (Ruegg etal., 1992, Neuron 8 691-699; Tsim et. al., supra). The EC₅₀ for theability to induce proliferation was 5 nM for the most active agonistclone when expressed as either scFv or IgG. The affinity of these scFvand IgG for MuSK was determined using BIAcore™ analysis. The agonistscFv and several non-agonist scFv each displayed affinity for MuSKwithin the range of 5-25 nM. In contrast, the affinities of the IgG forMuSK were 10-30 pM . See Table 5 below.

TABLE 5 clone # Agonist k_(d). k_(a) Affinity musk #2-scFv + 3.34 × 10⁻³8.78 × 10⁵ 3.8 nM musk #3-scFv − 2.39 × 10⁻³ 1.05 × 10⁵ 23 nM musk#4-scFv + 1.57 × 10⁻³ 1.84 × 10⁵ 8.5 nM musk #5-scFv − 2.49 × 10⁻³ 5.29× 10⁵ 4.7 nM musk #6-scFv − 4.95 × 10⁻³ 1.05 × 10⁵ 4.7 nM musk#13-scFv + 2.32 × 10⁻³ 4.53 × 10⁵ 5.1 nM musk #22-scFv + 6.09 × 10⁻³1.27 × 10⁵ 4.8 nM musk #13-IgG + 1.01 × 10⁻⁵ 8.05 × 10⁵ 12.5 pM musk#22-IgG + 4.86 × 10⁻⁵ 1.65 × 10⁶ 29.5 pM

To probe this agonist activity further, scFv were examined for theability to induce tyrosine phosphorylation of full length MuSK tyrosinekinase. The murine myoblastic cell line C2C12 was cultured underconditions that promote myotube differentiation and subsequently exposedto scFv, IgG or c-agrin. In correspondence with previous data (Glass et.al., supra), c-agrin was able to induce MuSK tyrosine phosphorylation.The agonist scFv and IgG were also found to rapidly induce tyrosinephosphorylation of MuSK as determined by western blot analysis withanti-phosphotyrosine antibody whereas other scFv and non-agonistanti-MuSK IgG were without effect.

The ability of the scFv MuSK agonists to induce AChR clustering incultured C2C12 myotubes was examined. Following stimulation, the cellswere fixed and the distribution of cell surface AChR was revealed withrhodamine labeled bungarotoxin. In undifferentiated myoblasts, AChR weredispersed and unfocused in the presence of c-agrin, scFv, or IgG. Incontrast, upon myotube differentiation, c-agrin and agonist scFv and IgGinduced marked aggregation of AChR into large and intensely stainedclusters. Non-agonist scFv and non-agonist IgG directed against MuSK oran irrelevant antigen were without effect. An additional consequence ofagrin action, tyrosine phosphorylation of subunits of the AChR was alsoexamined utilizing an antisera that recognizes the and chains of thereceptor. Tyrosine phosphorylation levels of both the and chains weremarkedly induced by c-agrin as well as the agonist scFv and agonist IgGbut were unaffected by control scFv and IgG.

Variants of the MuSK agonist antibodies of the invention may be preparedas described above for thrombopoietic antibodies.

Construction of expression vectors. Coding sequence for murine MuSK wasobtained by PCR amplification. MuSK-Fc was prepared by fusion of theextracellular domain of MuSK (a.a. 1-492) in frame with the Fc region ofhuman IgG1 in the eukaryotic expression vector pRK5tkNEO. MuSK-Fc wastrantsiently expressed in 293 cells and purified over a protein Gcolumn. A chimeric receptor, MuSK-Mpl, comprised of the extracellulardomain of MuSK (amino acids 1-492) and the transmembrane andintracellular domain of the human c-Mpl receptor (amino acids 491-635)was prepared by sequential PCR and cloned into pRK5tkNEO. Stable celllines expressing the chimeric receptor were obtained by electroporation(5 million cells, 250 volts, 960 μF) of linearized vector (20 μg) intoBa/F3 cells followed by selection for neomycin resistance with 2 mg/mlG418. Full length MuSK in pRK5tkNEO was transfected into 293 cells andstable transformants were obtained following two weeks of G418 selection(400 μg/ml). The sequence of the DNA constructs were confirmed by DNAsequencing. Expression of MuSK was assessed by flow cytometry analysisas described below. Ba/F3 cells were maintained in RPMI 1640 mediasupplemented with 10% fetal calf serum and 5% conditioned media fromWEH1-3B cells as source of IL-3. C-agrin (amino acids 1137-1949 of therat agrin (Ag+8 active splice form (Ferns et al, 1993, Neuron 11491-502.)) was expressed by transient transfection from 293 cells inserum free media with an expression vector, pRK-gD-c-Agrin, as a fusionprotein with the gD signal sequence and epitope tag and a genenasecleavage site (MGGAAARLGAVILFVVIVGLHGVRGKYALADASLKMADPNRFRGKDLPVLDQLLEGGAAHYALLPG) (SEQ ID NO. 71)fused to the N-terminus.

Isolation of scFv and IgG MuSK-Fc imiunoadhesin was coated on Maxisorptubes (Nunc) at 10 μg/ml. A library of human scFv (Cambridge AntibodyTechnology, England) was panned through two rounds of enrichmentessentially as described (Griffiths et al, 199, EMBO-J 12 725-734). Thespecificity of individual clones was assessed first by elisa (Griffithset al, supra) using MuSK-Fc and a control immunoadhesin (CD4-Fc).Positive clones were screened by PCR and “fingerprinted” by BstNIdigestion (Clackson et al, 1991, Nature 352 642-648.). Examples ofclones with unique patterns were sequenced and subjected to FACSanalysis with cells expressing or not expressing MuSK. For FACSanalysis, cells (10⁵) were incubated for 60 minutes at 4 C in 200 μl 2%FBS/PBS (fetal bovine serum/phosphate buffered saline) with 10¹⁰ phagethat were first blocked by incubation in 30 μl 10% FBS/PBS. Cells werethen washed with 2% FBS/PBS, stained with anti-M13 antibody (Pharmacia,Piscataway N.J.) and R-phycoerytherin-conjugated donkey anti-sheepantibody (Jackson Immunoresearch, West Grove Pa.), and analyzed by FACSanalysis. ScFv were expressed in bacteria as epitope tagged proteinscontaining a c-myc tag sequence recognized by monoclonal antibody 9E10(Griffiths et al, supra) and a polyhistidine tail (his₆) and werepurified over Ni-NTA column with imidazole elution as recommended bymanufacturer (Qiagen). For expression of clones as IgG the sequencesencoding the V_(H) and V_(L) regions of the scFv were introduced by PCRinto mammalian expression vector pIgG-kappa which was designed to enablethe expression of fully human light and heavy chains of kappa type IgG.Expression vectors for the individual clones were transfected into CHOcells and IgG were harvested from conditioned serum free media andpurified over a protein A column.

Proliferation assays. Cells were cultured in the absence of IL-3 fortwenty-two hours (in RPMI supplemented with 10% FBS). Cells were thenwashed twice with RPMI and plated in 96 well dishes at 50,000 cells perwell in 0.2 ml of 7.5% FBS RPMI supplemented with the indicatedconcentrations of scFv or IgG. Each concentration was tested induplicate. After an incubation of sixteen hours, 1 μCi of [³H]-thymidinewas added per well and incubation was continued for an additional sixhours. Incorporation of [³H]-thymidine was measured with a Top CountCounter (Packard Instruments, Calif.).

AChR clustering assay. C2C12 were maintained in 10% FBS in high glucoseDMEM at subfluency. For AChR clustering assays C2C12 were seeded onglass slides coated with fibronectin and poly-lysine and myotubedifferentiation was induced by 48 hour incubation in 2% horse serum highglucose DMEM. scFv or c-agrin were added to the culture medium andincubated overnight (16 hours). Cells were then washed with PBS andfixed in 4% paraformaldehyde. Rhodamine conjugated bungarotoxin(Molecular Probes, Eugene Oreg.) was used to reveal the localization ofAChRs as described (Ferns et al, supra).

Binding affinity analysis. Protein interaction analysis using BIAcoreTMinstruments was performed as described (Mark et al, 1996, J. Biol. Chem.271 9785-9789). Briefly, research grade CM5 sensor chips were activatedby injection of 20 μl of 1:1N-Ethyl-N′-(3-dimethylaminopropyl)carboiimide hydrochloride andN-hydroxysuccinimide at 5 μl/min flow rate. 20 μl of MuSK-Fc at 20μg/min in 10 mM sodium acetate, pH 5.0 was injected over the sensorchip, followed by 30 μl of ethanolamine. scFv or IgG were purified andconcentrations determined by Pearce BCA kit. Thirty μl protein samplesin PBS with 0.05% Tween 20 were injected at a flow rate of 10 μl min bythe Kinject method. Proteins were allowed to dissociate for 20 min in aflow of PBS with 0.05% Tween 20. Sensorgrams were analyzed withBIAevaluation 2.1 software from Pharmacia Biosensor AB. Apparentdissociation rate constants (k_(d)) and association rate constants(k_(a)) were obtained by evaluating the sensorgram with A+B=AB type Ifitting. Equilibrium dissociation constant K_(d) was calculated ask_(d)/k_(a).

Immunoprecipitation and Western Blot Analysis. C2C12 were maintained in10% FBS high glucose DMEM and induced to differentiate by 72 hourincubation in 2% horse serum. Cells were then stimulated by addition ofc-agrin, scFv or IgG for the time indicated in the figures. ScFv and IgGwere used at 50 nM. The c-agrin containing conditioned media was used atlevel that provided maximal tyrosine phosphorylation. Cell extracts wereprepared as described (Gurney et al, supra). Extracts were incubated for60 minutes at 4° C. with 30 μl agarose conjugated antiphosphotyrosinemonoclonal antibody 4G10 (UBI inc., Lake Placid N.Y.) or 1 μg ofanti-MuSK IgG #13 followed by 30 μl protein A sepharose beads. Westernblot analysis with antiphosphotyrosine antibody 4G10 or anti AChRantibody (Affinity Bioreagents, Golden Colo.) was performed ore asrecommended by the manufacturer and revealed with HRP conjugatedsecondary antibody and ECL (Amersham).

MuSK scFv are readily observed as dimers when resolved by nondenaturinggel electrophoresis. Additionally, the abundance of dimeric species maybe significantly altered in the local context of scFv bound to receptoron the cell surface. Alternatively, screening an scFv phage library witha divalent antigen, in this case MuSK-Fc, allows direction selection ofscFv that bind to and facilitate the formation of a receptor dimer.

While the invention has necessarily been described in conjunction withpreferred embodiments and specific working examples, one of ordinaryskill, after reading the foregoing specification, will be able to effectvarious changes, substitutions of equivalents, and alterations to thesubject matter set forth herein, without departing from the spirit andscope thereof. Hence, the invention can be practiced in ways other thanthose specifically described herein. It is therefore intended that theprotection granted by letters patent hereon be limited only by theappended claims and equivalents thereof.

All references cited herein are hereby expressly incorporated byreference.

Deposit of Material

The following materials have been deposited with the American TypeCulture Collection, 10801 University Boulecard, Manassas, Va., USA(ATCC):

Material ATCC Dep. No. Deposit Date pMpl.12B5.scFv.his      Aug. 18,1998 pMpl.12D5.scFv.his      Aug. 18, 1998 pMpl.12E10.scFv.his      Aug.18, 1998 pMpl.10D10.scFv.his      Aug. 18, 1998 pMpl.10F6.scFv.his     Aug. 18, 1998 pMpl.5E5.scFv.his      Aug. 18, 1998

This deposit was made under the provisions of the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure and the Regulations thereunder (BudapestTreaty). This assures maintenance of a viable culture of the deposit forat least 30 years and at least 5 years after the most recent request forthe furnishing of a sample of the deposit was received by the depositoryfrom the date of deposit. The deposit will be made available by ATCCunder the terms of the Budapest Treaty, and subject to an agreementbetween Genentech, Inc. and ATCC, which assures permanent andunrestricted availability of the progeny of the culture of the depositto the public upon issuance of the pertinent U.S. patent or upon layingopen to the public of any U.S. or foreign patent application, whichevercomes first, and assures availability of the progeny to one determinedby the U.S. Commissioner of Patents and Trademarks to be entitledthereto according to 35 USC §122 and the Commissioner's rules pursuantthereto (including 37 CFR §1.14 with particular reference to 886 OG638).

The assignee of the present application has agreed that if a culture ofthe materials on deposit should die or be lost or destroyed whencultivated under suitable conditions, the materials will be promptlyreplaced on notification with another of the same. Availability of thedeposited material is not to be construed as a license to practice theinvention in contravention of the rights granted under the authority ofany government in accordance with its patent laws.

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by the construct deposited,since the deposited embodiment is intended as a single illustration ofcertain aspects of the invention and any constructs that arefunctionally equivalent are within the scope of this invention. Thedeposit of material herein does not constitute an admission that thewritten description herein contained is inadequate to enable thepractice of any aspect of the invention, including the best modethereof, nor is it to be construed as limiting the scope of the claimsto the specific illustrations that it represents. Indeed, variousmodifications of the invention in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and fall within the scope of the appended claims.

77 1 15 DNA Homo sapiens 1 acc tct tgg atc ggc 15 Thr Ser Trp Ile Gly 15 2 5 PRT Homo sapiens 2 Thr Ser Trp Ile Gly 1 5 3 66 DNA Homo sapiens 3atc atg tat cct ggg aac tct gat acc aga cac aac 36 Ile Met Tyr Pro GlyAsn Ser Asp Thr Arg His Asn 1 5 10 ccg tcc ttc gaa gac cag gtc acc atgtca 66 Pro Ser Phe Glu Asp Gln Val Thr Met Ser 15 20 22 4 22 PRT Homosapiens 4 Ile Met Tyr Pro Gly Asn Ser Asp Thr Arg His Asn Pro Ser Phe 15 10 15 Glu Asp Gln Val Thr Met Ser 20 5 30 DNA Homo sapiens 5 gct ggggtc gcg ggc ggt gct ttt gat ctc 30 Ala Gly Val Ala Gly Gly Ala Phe AspLeu 1 5 10 6 10 PRT Homo sapiens 6 Ala Gly Val Ala Gly Gly Ala Phe AspLeu 1 5 10 7 42 DNA Homo sapiens 7 act gga acc agc agt ggc gtt ggt ggttat aac tat 36 Thr Gly Thr Ser Ser Gly Val Gly Gly Tyr Asn Tyr 1 5 10gtc tcc 42 Val Ser 14 8 14 PRT Homo sapiens 8 Thr Gly Thr Ser Ser GlyVal Gly Gly Tyr Asn Tyr Val Ser 1 5 10 9 21 DNA Homo sapiens 9 ggt aacagc aat cgg ccc tca 21 Gly Asn Ser Asn Arg Pro Ser 1 5 7 10 7 PRT Homosapiens 10 Gly Asn Ser Asn Arg Pro Ser 1 5 11 30 DNA Homo sapiens 11 agcaca tat gca ccc ccc ggt att att atg 30 Ser Thr Tyr Ala Pro Pro Gly IleIle Met 1 5 10 12 10 PRT Homo sapiens 12 Ser Thr Tyr Ala Pro Pro Gly IleIle Met 1 5 10 13 15 DNA Homo sapiens 13 gac tac tac atg agc 15 Asp TyrTyr Met Ser 1 5 14 5 PRT Homo sapiens 14 Asp Tyr Tyr Met Ser 1 5 15 66DNA Homo sapiens 15 tac att agt agt agt ggt agt acc ata tac tac gca 36Tyr Ile Ser Ser Ser Gly Ser Thr Ile Tyr Tyr Ala 1 5 10 gac tct gtg aagggc cga ttc acc atc tcc 66 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser 15 2022 16 22 PRT Homo sapiens 16 Tyr Ile Ser Ser Ser Gly Ser Thr Ile Tyr TyrAla Asp Ser Val 1 5 10 15 Lys Gly Arg Phe Thr Ile Ser 20 17 27 DNA Homosapiens 17 tgg agt ggt gag gat gct ttt gat atc 27 Trp Ser Gly Glu AspAla Phe Asp Ile 1 5 9 18 9 PRT Homo sapiens 18 Trp Ser Gly Glu Asp AlaPhe Asp Ile 1 5 19 33 DNA Homo sapiens 19 cgg gcc agt gag ggt att tatcac tgg ttg gcc 33 Arg Ala Ser Glu Gly Ile Tyr His Trp Leu Ala 1 5 10 2011 PRT Homo sapiens 20 Arg Ala Ser Glu Gly Ile Tyr His Trp Leu Ala 1 510 21 21 DNA Homo sapiens 21 aag gcc tct agt tta gcc agt 21 Lys Ala SerSer Leu Ala Ser 1 5 22 7 PRT Homo sapiens 22 Lys Ala Ser Ser Leu Ala Ser1 5 23 27 DNA Homo sapiens 23 caa caa tat agt aat tat ccg ctc act 27 GlnGln Tyr Ser Asn Tyr Pro Leu Thr 1 5 24 9 PRT Homo sapiens 24 Gln Gln TyrSer Asn Tyr Pro Leu Thr 1 5 25 15 DNA Homo sapiens 25 acc tac ggc atgcac 15 Thr Tyr Gly Met His 1 5 26 5 PRT Homo sapiens 26 Thr Tyr Gly MetHis 1 5 27 66 DNA Homo sapiens 27 ggt ata tcc ttt gac gga aga agt gaatac tat gca 36 Gly Ile Ser Phe Asp Gly Arg Ser Glu Tyr Tyr Ala 1 5 10gac tcc gtg aag ggc cga ttc acc atc tcc 66 Asp Ser Val Lys Gly Arg PheThr Ile Ser 15 20 28 22 PRT Homo sapiens 28 Gly Ile Ser Phe Asp Gly ArgSer Glu Tyr Tyr Ala Asp Ser Val 1 5 10 15 Lys Gly Arg Phe Thr Ile Ser 2029 27 DNA Homo sapiens 29 gat agg ggg tcc tac ggt atg gac gtc 27 Asp ArgGly Ser Tyr Gly Met Asp Val 1 5 30 9 PRT Homo sapiens 30 Asp Arg Gly SerTyr Gly Met Asp Val 1 5 31 66 DNA Homo sapiens 31 ggt ata tcc ttt gacgga aga agt gaa tac tat gca 36 Gly Ile Ser Phe Asp Gly Arg Ser Glu TyrTyr Ala 1 5 10 gac tcc gtg cag ggc cga ttc acc atc tcc 66 Asp Ser ValGln Gly Arg Phe Thr Ile Ser 15 20 22 32 22 PRT Homo sapiens 32 Gly IleSer Phe Asp Gly Arg Ser Glu Tyr Tyr Ala Asp Ser Val 1 5 10 15 Gln GlyArg Phe Thr Ile Ser 20 33 24 DNA Homo sapiens 33 gga gca cat tat ggt ttcgat atc 24 Gly Ala His Tyr Gly Phe Asp Ile 1 5 34 8 PRT homo sapiens 34Gly Ala His Tyr Gly Phe Asp Ile 1 5 35 33 DNA Homo sapiens 35 cgg gccagc gag ggt att tat cac tgg ttg gcc 33 Arg Ala Ser Glu Gly Ile Tyr HisTrp Leu Ala 1 5 10 36 15 DNA Homo sapiens 36 agc cat aac atg aac 15 SerHis Asn Met Asn 1 5 37 5 PRT Homo sapiens 37 Ser His Asn Met Asn 1 5 3866 DNA Homo sapiens 38 tcc att agt agt agt agt agt tac ata tac tac gca36 Ser Ile Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala 1 5 10 gac tca gtgaag ggc cga ttc acc atc tcc 66 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser15 20 39 22 PRT Homo sapiens 39 Ser Ile Ser Ser Ser Ser Ser Tyr Ile TyrTyr Ala Asp Ser Val 1 5 10 15 Lys Gly Arg Phe Thr Ile Ser 20 40 27 DNAHomo sapiens 40 gat cgc ggg agt acc ggt atg gac gtc 27 Asp Arg Gly SerThr Gly Met Asp Val 1 5 41 9 PRT Homo sapiens 41 Asp Arg Gly Ser Thr GlyMet Asp Val 1 5 42 15 DNA Homo sapiens 42 agt tac tac tgg agc 15 Ser TyrTyr Trp Ser 1 5 43 5 PRT Homo sapiens 43 Ser Tyr Tyr Trp Ser 1 5 44 63DNA Homo sapiens 44 tat atc tat tac agt ggg agc acc aac tac aac ccc 36Tyr Ile Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro 1 5 10 tcc ctc aag agtcga gtc acc ata tca 63 Ser Leu Lys Ser Arg Val Thr Ile Ser 15 20 45 21PRT Homo sapiens 45 Tyr Ile Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro SerLeu Lys 1 5 10 15 Ser Arg Val Thr Ile Ser 20 46 18 DNA Homo sapiens 46ggg agg tat ttt gac gtc 18 Gly Arg Tyr Phe Asp Val 1 5 47 6 PRT Homosapiens 47 Gly Arg Tyr Phe Asp Val 1 5 48 42 DNA Homo sapiens 48 act ggaacc agc agt gac gtt ggt ggt tat aac tat 36 Thr Gly Thr Ser Ser Asp ValGly Gly Tyr Asn Tyr 1 5 10 gtc tcc 42 Val Ser 14 49 14 PRT Homo sapiens49 Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr Asn Tyr Val Ser 1 5 10 50 21DNA Homo sapiens 50 gag ggc agt aag cgg ccc tca 21 Glu Gly Ser Lys ArgPro Ser 1 5 51 7 PRT Homo sapiens 51 Glu Gly Ser Lys Arg Pro Ser 1 5 5230 DNA Homo sapiens 52 agc tca tat aca acc agg agc act cga gtt 30 SerSer Tyr Thr Thr Arg Ser Thr Arg Val 1 5 10 53 10 PRT Homo sapiens 53 SerSer Tyr Thr Thr Arg Ser Thr Arg Val 1 5 10 54 23 DNA Artificial SequencePCR primer 54 agcggataac aatttcacac agg 23 55 21 DNA Artificial SequencePCR primer 55 gtcgtctttc cagacggtag t 21 56 44 PRT Artificial SequenceFab′2 antibody fragment 56 Cys Pro Pro Cys Ala Pro Glu Leu Leu Gly GlyArg Met Lys Gln 1 5 10 15 Leu Glu Asp Lys Val Glu Glu Leu Leu Ser LysAsn Tyr His Leu 20 25 30 Glu Asn Glu Val Ala Arg Leu Lys Lys Leu Val GlyGlu Arg 35 40 57 43 DNA Artificial Sequence PCR primer 57 gcttctgcggccacacaggc ctacgctgac atcgtgatga ccc 43 58 40 DNA Artificial SequencePCR primer 58 atgatgatgt gccacggtcc gtttgatctc cagttcggtc 40 59 43 DNAArtificial Sequence PCR primer 59 gcttctgcgg ccacacaggc ctacgcttcctatgtgctga ctc 43 60 40 DNA Artificial Sequence PCR primer 60 ccttctctctttaggttggc caaggacggt cagcttggtc 40 61 43 DNA Artificial Sequence PCRprimer 61 gcttctgcgg ccacacaggc ctacgctcag tctgtgctga ctc 43 62 39 DNAArtificial Sequence PCR primer 62 cattctacaa acgcgtacgc tcaggtgcagctggtgcag 39 63 45 DNA Artificial Sequence PCR primer 63 gtaaatgtatgggcccttgg tggaggaggc actcgagacg gtgac 45 64 39 DNA Artificial SequencePCR primer 64 cattctacaa acgcgtacgc tcaggtgcag ctggtggag 39 65 39 DNAArtificial Sequence PCR primer 65 cattctacaa acgcgtacgc tgacgtgcagctggtgcag 39 66 42 DNA Artificial Sequence PCR primer 66 gtaaatgtatgggcccttgg tggcggctga ggagacggtg ac 42 67 39 DNA Artificial Sequence PCRprimer 67 cattctacaa acgcgtacgc tcaggtgcag ctgcagcag 39 68 39 DNAArtificial Sequence PCR primer 68 cattctacaa acgcgtacgc tcaggtgcagctgcaggag 39 69 42 DNA Artificial Sequence PCR primer 69 gtaaatgtatgggcccttgg tggaggctga agagacggta ac 42 70 12 PRT Artificial Sequence gDtag 70 Met Ala Asp Pro Asn Arg Phe Arg Gly Lys Asp Leu 1 5 10 71 66 PRTArtificial Sequence Partial fusion protein sequence 71 Met Gly Gly AlaAla Ala Arg Leu Gly Ala Val Ile Leu Phe Val 1 5 10 15 Val Ile Val GlyLeu His Gly Val Arg Gly Lys Tyr Ala Leu Ala 20 25 30 Asp Ala Ser Leu LysMet Ala Asp Pro Asn Arg Phe Arg Gly Lys 35 40 45 Asp Leu Pro Val Leu AspGln Leu Leu Glu Gly Gly Ala Ala His 50 55 60 Tyr Ala Leu Leu Pro Gly 6572 249 PRT Artificial Sequence single chain antibody (scFv) fragments 72Met Ala Gln Val Gln Leu Gln Glu Ser Gly Gly Glu Met Lys Lys Pro 1 5 1015 Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly Tyr Gly Tyr Ser Phe Ala 20 2530 Thr Ser Trp Ile Gly Trp Val Arg Gln Met Pro Gly Arg Gly Leu Glu 35 4045 Trp Met Ala Ile Met Tyr Pro Gly Asn Ser Asp Thr Arg His Asn Pro 50 5560 Ser Phe Glu Asp Gln Val Thr Met Ser Ala Asp Thr Ser Ile Asn Thr 65 7075 80 Ala Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr 8590 95 Tyr Cys Ala Arg Ala Gly Val Ala Gly Gly Ala Phe Asp Leu Trp Gly100 105 110 Lys Gly Thr Met Val Thr Val Ser Ser Gly Gly Gly Gly Ser GlyGly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Gln Ser Val Leu Thr GlnPro Ala 130 135 140 Ser Val Ser Gly Ser Pro Gly Gln Ser Ile Thr Ile SerCys Thr Gly 145 150 155 160 Thr Ser Ser Gly Val Gly Gly Tyr Asn Tyr ValSer Trp Tyr Gln Gln 165 170 175 His Pro Gly Lys Ala Pro Lys Leu Leu IleTyr Gly Asn Ser Asn Arg 180 185 190 Pro Ser Gly Val Pro Asp Arg Phe SerAla Ser Lys Ser Gly Asn Thr 195 200 205 Ala Ser Leu Thr Ile Ser Gly LeuGln Ala Glu Asp Glu Ala Asp Tyr 210 215 220 Phe Cys Ser Thr Tyr Ala ProPro Gly Ile Ile Met Phe Gly Gly Gly 225 230 235 240 Thr Lys Leu Thr ValLeu Gly Ala Ala 245 73 245 PRT Artificial Sequence single chain antibody(scFv) fragments 73 Met Ala Glu Val Gln Leu Val Gln Ser Gly Gly Gly LeuVal Lys Pro 1 5 10 15 Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser GlyPhe Thr Phe Ser 20 25 30 Asp Tyr Tyr Met Ser Trp Ile Arg Gln Ala Pro GlyLys Gly Leu Glu 35 40 45 Trp Val Ser Tyr Ile Ser Ser Ser Gly Ser Thr IleTyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp AsnSer Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala GluAsp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Trp Ser Gly Glu Asp Ala PheAsp Ile Trp Gly Gln 100 105 110 Gly Thr Met Val Thr Val Ser Ser Gly GlyGly Gly Ser Gly Gly Gly 115 120 125 Gly Ser Gly Gly Gly Gly Ser Asp IleVal Met Thr Gln Ser Pro Ser 130 135 140 Thr Leu Ser Ala Ser Val Gly AspArg Val Ala Ile Thr Cys Arg Ala 145 150 155 160 Ser Glu Gly Ile Tyr HisTrp Leu Ala Trp Tyr Gln Gln Lys Pro Gly 165 170 175 Lys Ala Pro Lys LeuLeu Ile Tyr Lys Ala Ser Ser Leu Ala Ser Gly 180 185 190 Ala Pro Ser ArgPhe Ser Gly Ser Gly Ser Gly Ala Asp Phe Thr Leu 195 200 205 Thr Ile SerSer Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gln 210 215 220 Gln TyrSer Asn Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu 225 230 235 240Val Lys Arg Ala Ala 245 74 245 PRT Artificial Sequence single chainantibody (scFv) fragments 74 Met Ala Glu Val Gln Leu Val Gln Ser Gly GlyGly Val Val Gln Pro 1 5 10 15 Gly Gly Ser Leu Ser Leu Ser Cys Ala ValSer Gly Ile Thr Leu Arg 20 25 30 Thr Tyr Gly Met His Trp Val Arg Gln AlaPro Gly Lys Gly Leu Glu 35 40 45 Trp Val Ala Gly Ile Ser Phe Asp Gly ArgSer Glu Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser ArgAsp Asn Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu ArgAla Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Asp Arg Gly Ser TyrGly Met Asp Val Trp Gly Arg 100 105 110 Gly Thr Met Val Thr Val Ser SerGly Gly Gly Gly Ser Gly Gly Gly 115 120 125 Gly Ser Gly Gly Gly Gly SerAsp Ile Gln Met Thr Gln Ser Pro Ser 130 135 140 Thr Leu Ser Ala Ser IleGly Asp Arg Val Thr Ile Thr Cys Arg Ala 145 150 155 160 Ser Glu Gly IleTyr His Trp Leu Ala Trp Tyr Gln Gln Lys Pro Gly 165 170 175 Lys Ala ProLys Leu Leu Ile Tyr Lys Ala Ser Ser Leu Ala Ser Gly 180 185 190 Ala ProSer Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu 195 200 205 ThrIle Ser Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gln 210 215 220Gln Tyr Ser Asn Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu 225 230235 240 Ile Leu Arg Ala Ala 245 75 244 PRT Artificial Sequence singlechain antibody (scFv) fragments 75 Met Ala Gln Val Gln Leu Val Gln SerGly Gly Gly Leu Val Arg Pro 1 5 10 15 Gly Gly Ser Leu Ser Leu Ser CysAla Val Ser Gly Ile Thr Leu Arg 20 25 30 Thr Tyr Gly Met His Trp Val ArgGln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp Val Ala Gly Ile Ser Phe AspGly Arg Ser Glu Tyr Tyr Ala Asp 50 55 60 Ser Val Gln Gly Arg Phe Thr IleSer Arg Asp Ser Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn SerLeu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Gly Ala HisTyr Gly Phe Asp Ile Trp Gly Gln Gly 100 105 110 Thr Met Val Thr Val SerSer Gly Gly Gly Gly Thr Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly GlySer Asp Ile Gln Met Thr Gln Ser Pro Ser Thr 130 135 140 Leu Ser Ala SerIle Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser 145 150 155 160 Glu GlyIle Tyr His Trp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys 165 170 175 AlaPro Lys Leu Leu Ile Tyr Lys Ala Ser Ser Leu Ala Ser Gly Ala 180 185 190Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 195 200205 Ile Ser Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 210215 220 Tyr Ser Asn Tyr Pro Leu Thr Phe Gly Gly Gly Thr Glu Leu Glu Ile225 230 235 240 Lys Arg Ala Ala 76 245 PRT Artificial Sequence singlechain antibody (scFv) fragments 76 Met Ala Gln Val Gln Leu Val Glu SerGly Gly Gly Leu Val Lys Pro 1 5 10 15 Gly Gly Ser Leu Arg Leu Ser CysAla Ala Ser Gly Phe Thr Phe Ser 20 25 30 Ser His Asn Met Asn Trp Val ArgGln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp Val Ser Ser Ile Ser Ser SerSer Ser Tyr Ile Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr IleSer Arg Asp Asn Ala Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn SerLeu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Asp Arg GlySer Thr Gly Met Asp Val Trp Gly Arg 100 105 110 Gly Thr Leu Val Thr ValSer Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125 Gly Ser Gly Gly GlyGly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser 130 135 140 Thr Leu Ser AlaSer Ile Gly Asp Arg Val Thr Ile Thr Cys Arg Ala 145 150 155 160 Ser GluGly Ile Tyr His Trp Leu Ala Trp Tyr Gln Gln Lys Pro Gly 165 170 175 LysAla Pro Lys Leu Leu Ile Tyr Lys Ala Ser Ser Leu Ala Ser Gly 180 185 190Ala Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Xaa 195 200205 Thr Ile Ser Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gln 210215 220 Gln Tyr Ser Asn Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu225 230 235 240 Ile Lys Arg Ala Ala 245 77 244 PRT Artificial Sequencesingle chain antibody (scFv) fragments 77 Met Ala Gln Val Gln Leu GlnGln Ser Gly Pro Gly Leu Val Lys Pro 1 5 10 15 Ser Glu Thr Leu Ser LeuThr Cys Thr Val Ser Gly Asp Ser Ile Ser 20 25 30 Ser Tyr Tyr Trp Ser TrpIle Arg Gln Pro Pro Gly Lys Gly Leu Glu 35 40 45 Trp Ile Gly Tyr Ile TyrTyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Arg Val ThrIle Ser Val Asp Thr Ser Lys Ser Gln Phe 65 70 75 80 Ser Leu Lys Leu SerSer Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Gly ArgTyr Phe Asp Val Trp Gly Arg Gly Thr Met Val 100 105 110 Thr Val Ser SerGly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 115 120 125 Gly Gly SerSer Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ser 130 135 140 Pro GlyGln Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val 145 150 155 160Gly Gly Tyr Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala 165 170175 Pro Lys Leu Met Ile Tyr Glu Gly Ser Lys Arg Pro Ser Gly Val Ser 180185 190 Asn Arg Phe Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile195 200 205 Ser Gly Leu Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser SerTyr 210 215 220 Thr Thr Arg Ser Thr Arg Val Phe Gly Gly Gly Thr Lys LeuThr Val 225 230 235 240 Leu Gly Ala Ala

What is claimed is:
 1. An isolated nucleic acid encoding an agonistantibody, fragment or variant thereof which binds to human c-mpl,wherein said antibody, fragment or variant thereof is selected from thegroup consisting of: Ab1, Ab2, Ab3, Ab4, Ab5 and Ab6, wherein eachAb1-Ab6 comprises a VH and VL chain, each VH and VL chain comprising CDRamino acid sequences designated CDR1, CDR2 and CDR3 separated byframework amino acid sequences, the amino acid sequence of each CDR ineach VH and VL chain of Ab1-Ab6 is selected according to the followingtable: Ab1: VH^(CDR1) VH^(CDR2) VH^(CDR3) (SEQ ID NO: 1) (SEQ ID NO: 3)(SEQ ID NO: 5) (SEQ ID NO: 2) (SEQ ID NO: 4) (SEQ ID NO: 6) VL^(CDR1)VL^(CDR2) VL^(CDR3) (SEQ ID NO: 7) (SEQ ID NO: 9) (SEQ ID NO: 11) (SEQID NO: 8) (SEQ ID NO: 10) (SEQ ID NO: 12) Ab2: VH^(CDR1) VH^(CDR2)VH^(CDR3) (SEQ ID NO: 13) (SEQ ID NO: 15) (SEQ ID NO: 17) (SEQ ID NO:14) (SEQ ID NO: 16) (SEQ ID NO: 18) VL^(CDR1) VL^(CDR2) VL^(CDR3) (SEQID NO: 19) (SEQ ID NO: 21) (SEQ ID NO: 23) (SEQ ID NO: 20) (SEQ ID NO:22) (SEQ ID NO: 24) Ab3: VH^(CDR1) VH^(CDR2) VH^(CDR3) (SEQ ID NO: 25)(SEQ ID NO: 27) (SEQ ID NO: 29) (SEQ ID NO: 26) (SEQ ID NO: 28) (SEQ IDNO: 30) VL^(CDR1) VL^(CDR2) VL^(CDR3) (SEQ ID NO: 19) (SEQ ID NO: 21)(SEQ ID NO: 23) (SEQ ID NO: 20) (SEQ ID NO: 22) (SEQ ID NO: 24) Ab4:VH^(CDR1) VH^(CDR2) VH^(CDR3) (SEQ ID NO: 25) (SEQ ID NO: 31) (SEQ IDNO: 33) (SEQ ID NO: 26) (SEQ ID NO: 32) (SEQ ID NO: 34) VL^(CDR1)VL^(CDR2) VL^(CDR3) (SEQ ID NO: 35) (SEQ ID NO: 21) (SEQ ID NO: 23) (SEQID NO: 20) (SEQ ID NO: 22) (SEQ ID NO: 24) Ab5: VH^(CDR1) VH^(CDR2)VH^(CDR3) (SEQ ID NO: 36) (SEQ ID NO: 38) (SEQ ID NO: 40) (SEQ ID NO:37) (SEQ ID NO: 39) (SEQ ID NO: 41) VL^(CDR1) VL^(CDR2) VL^(CDR3) (SEQID NO: 19) (SEQ ID NO: 21) (SEQ ID NO: 23) (SEQ ID NO: 20) (SEQ ID NO:22) (SEQ ID NO: 24) Ab6: VH^(CDR1) VH^(CDR2) VH^(CDR3) (SEQ ID NO: 42)(SEQ ID NO: 44) (SEQ ID NO: 46) (SEQ ID NO: 43) (SEQ ID NO: 45) (SEQ IDNO: 47) VL^(CDR1) VL^(CDR2) VL^(CDR3) (SEQ ID NO: 48) (SEQ ID NO: 50)(SEQ ID NO: 52) (SEQ ID NO: 49) (SEQ ID NO: 51) (SEQ ID NO: 53).


2. A vector comprising the nucleic acid of claim
 1. 3. A host cellcomprising the vector of claim
 2. 4. A method of producing an agonistantibody comprising culturing the host cell of claim 3 under conditionswherein the nucleic acid is expressed.
 5. An isolated nucleic acidencoding an agonist antibody, fragment or variant thereof which binds tohuman c-mpl, wherein said agonist antibody, fragment, or variant thereofis selected from the group consisting of 12E10 (SEQ ID NO:77), 12B5 (SEQID NO:75), 10F6 (SEQ ID NO:72) and 12D5 (SEQ ID NO:76).
 6. The isolatednucleic acid of claim 1, wherein said agonist antibody, fragment, orvariant thereof is a humanized antibody, fragment or variant thereof. 7.The isolated nucleic acid of claim 1, wherein said agonist antibody,fragment, or variant thereof is a non-naturally occurring antibody,fragment or variant thereof.
 8. The isolated nucleic acid of claim 1,wherein said agonist antibody, fragment, or variant thereof is a humanantibody, fragment or variant thereof.
 9. The isolated nucleic acid ofclaim 1, wherein said agonist antibody stimulates proliferation,differentiation or growth of megakaryocytes.
 10. The isolated nucleicacid of claim 1, wherein said agonist antibody stimulates megakaryocytesto produce platelets.
 11. The antibody of claim 1, wherein said agonistantibody is selected from the group consisting of svFv, Fab, F(ab′)₂ andIgG.
 12. The antibody of claim 1, wherein said agonist antibody is amonoclonal antibody.